Semiochemical responsive olfactory sensory neurons are sexually dimorphic and plastic
Figures
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Identification of ORs exhibiting sexually dimorphic expression in sex-separated mice.
(A) Schematic of the housing setup. For sex-separation, male mice were co-housed exclusively with male mice. Female mice were co-housed exclusively with female mice. (B) Volcano plot comparing expression of Olfrs between 3-week-old sex-separated male and female mice. Olfr910, Olfr912, and Olfr1295 are highlighted in red. The red dashed line indicates an FDR = 0.05. Data are from n = 3 male and n = 3 female mice. (C) Volcano plot comparing expression of Olfrs between 9-week-old sex-separated male and female mice. (D) Volcano plot comparing expression of Olfrs between 26-week-old sex-separated male and female mice. (E) Volcano plot comparing expression of Olfrs between 43-week-old sex-separated male and female mice. (F) Longitudinal plotting of the mean and SEM of proportions of reads aligned to Olfr910, Olfr912, and Olfr1295 in sex-separated male and female mice.
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Sexually dimorphic expression of ORs is consistent with a change in the number of OSNs expressing those ORs.
(A) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr910 in 43-week-old sex-separated male (top) and female (bottom) mice. Scale bars indicate 50 μm. Right: summary data showing mean and SEM of the density of OSNs expressing Olfr910 in 43-week-old male and female mice. An unpaired two-tailed t-test revealed statistical difference (****p < 0.0001) between males and females. Data are from n = 3 male and n = 3 female mice. (B) Representative in situ mRNA hybridization pictures probing for the expression of Olfr912 in 43-week-old sex-separated male (top) and female (bottom) mice. Right: summary data showing mean and SEM of the density of OSNs expressing Olfr912 in 43-week-old male and female mice. An unpaired two-tailed t-test revealed statistical difference (****p < 0 .0001) between males and females. (C) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr1295 in 43-week-old sex-separated male (top) and female (bottom) mice. Right: summary data showing mean and SEM of the density of OSNs expressing Olfr1295 in 43-week-old male and female mice. An unpaired two-tailed t-test revealed statistical difference(****p < 0 .0001) between males and females.
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Figure 2—source data 1
Summary statistics for Figure 2.
- https://cdn.elifesciences.org/articles/54501/elife-54501-fig2-data1-v2.xlsx
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Architecture of reads uniquely mapping to female-enriched ORs.
(A) Architecture of reads uniquely mapped to Olfr910 in male and female mice. Shown are the defined transcript variants, coding sequence (CDS), uniquely designed riboprobe, and 5′ to 3′ directionality (red arrows). The y-axis shows the number of mapped reads along genomic coordinates described on the x-axis. Reads crossing exon junctions are also quantified. (B) Architecture of reads uniquely mapped to Olfr912. (C) Architecture of reads uniquely mapped to Olfr1295.
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Sexually dimorphic ORs are activated by mature male mouse odor.
(A) Schematic of exposure experiment. A juvenile mouse (black) was exposed to (in descending order) a clean environment, mature male mice, mature female mice, or 1% (v/v) acetophenone for 1 hr in a sealed container. (B) Left: representative in situ mRNA hybridization and pS6 immunostaining showing co-localization events, as indicated by arrowheads, between OSNs expressing Olfr910 and pS6 signal induction following exposure of a juvenile mouse to adult male mice. Scale bars indicate 20 μm. Right: summary data showing the mean and SEM of pS6 induction in OSNs expressing Olfr910 following exposure of a juvenile mouse to multiple stimuli. One-way ANOVA with Dunnett’s multiple comparisons test correction reveals that only exposure to mature male mice leads to significant (****p < 0.0001) pS6 induction within OSNs expressing Olfr910. Data are from n = 3 juvenile mice. (C) Left: representative in situ mRNA hybridization and pS6 immunostaining showing co-localization events between OSNs expressing Olfr912 and pS6 signal induction following exposure of a juvenile mouse to adult male mice. Right: summary data showing the mean and SEM of pS6 induction in OSNs expressing Olfr912 following exposure of a juvenile mouse to multiple stimuli (****p < 0.0001). (D) Left: representative in situ mRNA hybridization and pS6 immunostaining showing co-localization events between OSNs expressing Olfr1295 and pS6 signal induction following exposure of a juvenile mouse to adult male mice. Right: summary data showing the mean and SEM of pS6 induction in OSNs expressing Olfr1295 following exposure of a juvenile mouse to multiple stimuli (****p < 0.0001).
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Figure 3—source data 1
Summary statistics for Figure 3.
- https://cdn.elifesciences.org/articles/54501/elife-54501-fig3-data1-v2.xlsx
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Example in situ stainings showing sexually dimorphic ORs are activated by mature male mouse odor.
(A) Representative images showing in situ mRNA hybridizations probing for Olfr910 expression and pS6 immunostainings. Co-localization events are only seen following mature male mouse exposure as indicated by arrowheads. Scale bars indicate 20 μm. (B) Representative images showing in situ mRNA hybridizations probing for Olfr912 expression and pS6 immunostainings. (C) Representative images showing in situ mRNA hybridizations probing for Olfr1295 expression and pS6 immunostainings.
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Sexually dimorphic ORs are activated by mature male mouse semiochemicals SBT and MTMT.
(A) Schematic of the pS6-IP-Seq experiment. Litter matched, ~3 week-old (juvenile) mice are used. Mice are habituated to an odor-free environment for 1 hr. One mouse then receives exposure to an odor stimulus, while another receives exposure to the diluent, each for 1 hr. Whole olfactory mucosa is then harvested and immunoprecipitated using an antibody against pS6. (B) The panel of sex-specific and sex-enriched volatiles screened using pS6-IP-Seq. (C) Volcano plot showing the results of pS6-IP-Seq using 0.01% (v/v) SBT diluted in water as stimulus. Olfr910, Olfr912, and Olfr1295 are highlighted in red. The red dashed line indicates an FDR = 0.05. Data are from n = 3 control (diluent-exposed) mice and n = 3 experimental (odor-exposed) mice. (D) Volcano plot showing the results of pS6-IP-Seq using 100 μM MTMT dissolved in ethanol as stimulus. (E) Top: representative in situ mRNA hybridization and pS6 immunostaining showing co-localization events between OSNs expressing Olfr910 and pS6 signal induction following exposure of a juvenile mouse to 1% (v/v) SBT diluted in water. Scale bars indicate 20 μm. Bottom: summary data showing the mean and SEM of pS6 induction in OSNs expressing Olfr910 following exposure of a juvenile mouse to increasing concentrations of SBT and 1% (v/v) acetophenone. One-way ANOVA with Dunnett’s multiple comparisons test correction reveals only exposure to 0.01% (v/v) SBT, 0.1% (v/v) SBT, and 1% (v/v) SBT leads to significant pS6 induction within OSNs expressing Olfr910 (****p < 0.0001). Data are from n = 3 juvenile mice. (F) Top: representative in situ mRNA hybridization and pS6 immunostaining showing co-localization events between OSNs expressing Olfr912 and pS6 signal induction following exposure of a juvenile mouse to 1% (v/v) SBT diluted in water. Bottom: summary data showing the mean and SEM of pS6 induction in OSNs expressing Olfr912 following exposure of a juvenile mouse to increasing concentrations of SBT and 1% (v/v) acetophenone (**p < 0.01, ****p < 0.0001). (G) Top: representative in situ mRNA hybridization and pS6 immunostaining showing co-localization events between OSNs expressing Olfr1295 and pS6 signal induction following exposure of a juvenile mouse to 10 mM MTMT diluted in ethanol. Bottom: summary data showing the mean and SEM of pS6 induction in OSNs expressing Olfr1295 following exposure of a juvenile mouse to increasing concentrations of MTMT and 1% (v/v) acetophenone (****p < 0.0001).
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Figure 4—source data 1
Summary statistics for Figure 4.
- https://cdn.elifesciences.org/articles/54501/elife-54501-fig4-data1-v2.xlsx
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Sexually dimorphic ORs are not activated by sex-specific or sex-enriched odorants that are not SBT or MTMT.
(A) Volcano plot showing the results of pS6-IP-Seq using 1% (v/v) β-caryophyllene dissolved in water as stimulus. Olfr910, Olfr912, and Olfr1295 are highlighted in red and not enriched. The red dashed line indicates an FDR = 0.05. Data are from n = 3 control (diluent-exposed) mice and n = 3 experimental (odor-exposed) mice. (B) Volcano plot showing the results of pS6-IP-Seq using 1% (v/v) 2-heptanone dissolved in water as stimulus. (C) Volcano plot showing the results of pS6-IP-Seq using 100% (E)-β-farnesene dissolved in water as stimulus. (D) Volcano plot showing the results of pS6-IP-Seq using 77% (v/v) DHB dissolved in water as stimulus. (E) Volcano plot showing the results of pS6-IP-Seq using 1% (v/v) 2,5-DMP dissolved in water as stimulus. (F) Volcano plot showing the results of pS6-IP-Seq using 1% (v/v) SBT dissolved in water as stimulus. (G) Volcano plot showing the results of pS6-IP-Seq using 100% SBT as stimulus. (H) Volcano plot showing the results of pS6-IP-Seq using 10 mM MTMT dissolved in ethanol as stimulus.
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Cognate ORs for other sex-specific and sex-enriched volatiles are not sexually dimorphic.
(A) Left: the top five candidate ORs activated by 1% (v/v) 2-heptanone exposure, based on lowest FDR values, are highlighted in red. Right: none of the top five candidate ORs activated by 1% (v/v) 2-heptanone exposure exhibit sexual dimorphism in 43-week-old sex-separated mice. Data are from n = 3 control (diluent-exposed) mice and n = 3 experimental (odor-exposed) mice. (B) Left: the top five candidate ORs activated by 100% (E)-β-farnesene exposure, based on lowest FDR values, are highlighted in red. Right: none of the top five candidate ORs activated by 100% (E)-β-farnesene exposure exhibit sexual dimorphism in 43-week-old sex-separated mice. (C) Left: the top five candidate ORs activated by 77% (v/v) DHB exposure, based on lowest FDR values, are highlighted in red. Right: none of the top five candidate ORs activated by 77% (v/v) DHB exposure exhibit sexual dimorphism in 43-week-old sex-separated mice. (D) Left: the top five candidate ORs activated by 1% (v/v) 2,5-DMP exposure, based on lowest FDR values, are highlighted in red. Right: none of the top five candidate ORs activated by 1% (v/v) 2,5-DMP exposure exhibit sexual dimorphism in 43-week-old sex-separated mice.
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Example in situ stainings showing sexually dimorphic ORs are activated by SBT and MTMT.
(A) Representative images showing in situ mRNA hybridizations probing for Olfr910 expression and pS6 immunostainings. Co-localization events are only seen following SBT exposure as indicated by arrowheads. Scale bars indicate 20 μm. (B) Representative images showing in situ mRNA hybridizations probing for Olfr912 expression and pS6 immunostainings. (C) Representative images showing in situ mRNA hybridizations probing for Olfr1295 expression and pS6 immunostainings.
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OSN responses to semiochemicals are not sexually dimorphic between mature male and female mice.
(A) Comparison of responses of OSNs from 26-week-old male and female mice to various stimuli. One-way ANOVA with Tukey’s multiple comparisons test correction reveals only exposure to 0.1% (v/v) SBT leads to significant pS6 induction in OSNs expressing Olfr910 (***p < 0.001) with no significant differences between males and females (ns; p > 0.05). Data are from n = 3 male and n = 3 female mice. (B) One-way ANOVA with Tukey’s multiple comparisons test correction reveals only exposure to 0.1% (v/v) SBT leads to significant pS6 induction in OSNs expressing Olfr912 (**p < 0.01, ****p < 0.0001) with no significant differences between males and females (ns; p > 0.05). (C) One-way ANOVA with Tukey’s multiple comparisons test correction reveals only exposure to 10 mM MTMT leads to significant pS6 induction in OSNs expressing Olfr1295 (****p < 0.0001) with no significant differences between males and females (ns; p > 0.05).
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Figure 5—source data 1
Summary statistics for Figure 5.
- https://cdn.elifesciences.org/articles/54501/elife-54501-fig5-data1-v2.xlsx
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Example in situ stainings showing responses to semiochemicals are not sexually dimorphic between mature male and female mice.
(A) Representative images from mature male and female mice showing in situ mRNA hybridizations probing for Olfr910 expression and pS6 immunostainings. Co-localization events are only seen following SBT exposure as indicated by arrowheads. Scale bars indicate 20 μm. (B) Representative images from mature male and female mice showing in situ mRNA hybridizations probing for Olfr912 expression and pS6 immunostainings. Co-localization events are only seen following SBT exposure. (C) Representative images from mature male and female mice showing in situ mRNA hybridizations probing for Olfr1295 expression and pS6 immunostainings. Co-localization events are only seen following MTMT exposure.
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Sex-combined housing leads to the attenuation of the dimorphic OR representations.
(A) Schematic of the housing setup. For sex-combined housing, one male mouse was co-housed with one female mouse. (B) Volcano plot comparing expression of Olfrs between 43-week-old sex-combined male and female mice. Olfr910, Olfr912, and Olfr1295 are highlighted in red. The red dashed line indicates an FDR = 0.05. Data are from n = 3 male and n = 3 female mice. (C) Longitudinal plotting of the mean and SEM of proportions of reads aligned to Olfr910, Olfr912, and Olfr1295 in sex-separated and sex-combined male and female mice. (D) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr910 in 43-week-old sex-combined male (top) and female (bottom) mice. Scale bars indicate 50 μm. Right: summary data showing the mean and SEM of the density of OSNs expressing Olfr910 in 43-week-old male and female mice. One-way ANOVA with Tukey’s multiple comparisons test correction reveals only sex-separated female mice to differ in the density of OSNs expressing Olfr910 (****p < 0.0001). Data are from n = 3 male and n = 3 female mice from each housing condition. (E) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr912 in 43-week-old sex-combined male (top) and female (bottom) mice. Right: summary data showing the mean and SEM of the density of OSNs expressing Olfr912 in 43-week-old male and female mice. One-way ANOVA with Tukey’s multiple comparisons test correction reveals only sex-separated female mice to differ in the density of OSNs expressing Olfr912 (****p < 0.0001). (F) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr1295 in 43-week-old sex-combined male (top) and female (bottom) mice. Right: summary data showing the mean and SEM of the density of OSNs expressing Olfr1295 in 43-week-old male and female mice. One-way ANOVA with Tukey’s multiple comparisons test correction reveals only sex-separated female mice to differ in the density of OSNs expressing Olfr1295 (****p < 0.0001).
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Figure 6—source data 1
Summary statistics for Figure 6.
- https://cdn.elifesciences.org/articles/54501/elife-54501-fig6-data1-v2.xlsx
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Olfr1437 and Olfr235 are male-enriched and respond to the macrocyclic musk molecule CPD.
(A) Volcano plot comparing expression of Olfrs between 43-week-old sex-separated male and female mice. Olfr1437 and Olfr235 and are highlighted in red. The red dashed line indicates an FDR = 0.05. Data are from n = 3 male and n = 3 female mice. (B) Volcano plot comparing expression of Olfrs between 43-week-old sex-combined male and female mice. (C) Longitudinal plotting of the mean and SEM of proportions of reads aligned to Olfr1437 and Olfr235 in sex-separated and sex-combined male and female mice. (D) Structure of macrocyclic musk odorant cyclopentadecanone (CPD). (E) Volcano plot showing the results of pS6-IP-Seq using 100 mM CPD diluted in ethanol as stimulus. Olfr1437 and Olfr235 are highlighted in red and enriched. The red dashed line indicates an FDR = 0.05. Data are from n = 3 control (diluent-exposed) mice and n = 3 experimental (odor-exposed) mice.
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Male over-expression is also consistent with a difference in the number of cells expressing Olfr1437 between male and female mice.
(A) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr1437 in 43-week-old sex-separated male (top) and female (bottom) mice. Scale bars indicate 50 μm. Right: summary data showing mean and SEM of the density of OSNs expressing Olfr1437 in 43-week-old sex-separated male (top) and female (bottom) mice. An unpaired two-tailed t-test revealed statistical difference (****p < 0.0001) between males and females. Data are from n = 3 male and n = 3 female mice.
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Figure 7—figure supplement 1—source data 1
Summary statistics for Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/54501/elife-54501-fig7-figsupp1-data1-v2.xlsx
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Single-cell RNA-Seq analysis reveals an enrichment of activity-associated gene expression in male OSNs expressing Olfr910, Olfr912, and Olfr1295.
(A) Chemosensory receptor-independent UMAP embedding of 17,666 singly sequenced mature OSNs. OSNs expressing Olfr910, Olfr912, Olfr1295, Olfr1437, and Olfr235 are highlighted. (B) Gene expression analysis identified activity-associated genes S100a5, Ptprn, Pcp4l1, and Nrp1 to be enriched amongst OSNs expressing Olfr910, Olfr912, and Olfr1295. (C) UMAP representations of S100a5, Ptprn, Pcp4l1, and Nrp1 show a tendency toward higher expression where OSNs expressing Olfr910, Olfr912, and Olfr1295 are localized.
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Figure 8—source data 1
Summary statistics for Figure 8.
- https://cdn.elifesciences.org/articles/54501/elife-54501-fig8-data1-v2.xlsx
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Sex-separated Bax-/- mice fail to generate sexually dimorphic representations of Olfr910, Olfr912, and Olfr1295.
(A) Volcano plot comparing expression of Olfrs between 26-week-old Bax-/- sex separated male and female mice. Olfr910, Olfr912, and Olfr1295 are highlighted in red. The red dashed line indicates an FDR = 0.05. Data are from n = 3 male and n = 3 female mice. (B) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr910 in Bax-/- 43-week-old sex-separated male (top) and female (bottom) mice. Scale bars indicate 50 μm. Right: summary data showing mean and SEM of the density of OSNs expressing Olfr910 in Bax-/- 43 week-old sex-separated male and female mice. An unpaired two-tailed t-test reveals no statistical difference (ns; p > 0.05) between males and females. Data are from n = 3 male and n = 3 female mice. (C) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr912 in Bax-/- 43-week-old sex-separated male (top) and female (bottom) mice. Right: summary data showing mean and SEM of the density of OSNs expressing Olfr912 in Bax-/- 43-week-old male and female mice. An unpaired two-tailed t-test reveals no statistical difference between males and females (ns; p > 0.05). (D) Left: representative in situ mRNA hybridization pictures probing for the expression of Olfr1295 in Bax-/- 43-week-old sex-separated male (top) and female (bottom) mice. Right: summary data showing mean and SEM of the density of OSNs expressing Olfr1295 in Bax-/- 43-week-old male and female mice. An unpaired two-tailed t-test reveals no statistical difference between males and females (ns; p > 0.05).
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Figure 9—source data 1
Summary statistics for Figure 9.
- https://cdn.elifesciences.org/articles/54501/elife-54501-fig9-data1-v2.xlsx
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Sex-separated Bax-/- mice fail to generate sexually dimorphic representations of Olfr1437 and Olfr235. .
(A) Volcano plot comparing expression of Olfrs between 26-week-old Bax-/- sex-separated male and female mice. Olfr1437 and Olfr235 are highlighted in red. The red dashed line indicates an FDR = 0.05. Data are from n = 3 male and n = 3 female mice.
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Bax-/- mice exhibit significant changes in their OR repertoire.
(A) Volcano plot comparing expression of Olfrs between 26-week-old wild-type and Bax-/- sex-separated male mice. Olfr910, Olfr912, Olfr1295, Olfr1437, and Olfr235 are highlighted in red. The red dashed line indicates an FDR = 0.05. Data are from n = 3 wild-type and n = 3 mutant mice. (B) Volcano plot comparing expression of Olfrs between 26-week-old wild-type and Bax-/- sex-separated female mice.
Tables
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Mus musculus C57BL/6J) | C57BL/6J | Jackson Labs | 000664 | |
Strain, strain background (Mus musculus C57BL/6J) | Bax-/- | Jackson Labs | 002994 | |
Commercial assay, kit | TRIzol | Life Technologies | 15596026 | |
Commercial assay, kit | QUBIT HS RNA Assay Kit | ThermoFisher | Q32855 | |
Commercial assay, kit | RNase-free DNaseI | Roche | 04 716 728 001 | |
Commercial assay, kit | RNeasy Mini Kit | Qiagen | 74104 | |
Commercial assay, kit | SMART-Seq v4 Ultra Low Input RNA Kit | Takara | 634898 | |
Commercial assay, kit | Nexterra XT DNA Library Preparation Kit | Illumina | 15032354 | |
Sequence-based reagent | Olfr910 UTR F | This paper | 5'-AAACGCGTGTGAAAATTGTGACAGATCCA-3' | |
Sequence-based reagent | Olfr910 UTR R | This paper | 5'-AAGCGGCCGCCATTTACAAGAAGGGAATCAG-3' | |
Sequence-based reagent | Olfr912 UTR F | This paper | 5'-AAACGCGTACTTTGTTCTGATTCAGTTGTT-3' | |
Sequence-based reagent | Olfr912 UTR R | This paper | 5′-AAGCGGCCGCGTCCACAGAGCAATACAACA-3′ | |
Sequence-based reagent | Olfr1295 UTR F | This paper | 5'-AAACGCGTACTCCTCTCCTAAATCCAAC-3' | |
Sequence-based reagent | Olfr1295 UTR R | This paper | 5'-AAGCGGCCGCGGCAGCACCACTGATCAA-3' | |
Commercial assay, kit | Phusion DNA Polymerase | NEB | F530S | |
Commercial assay, kit | pCI vector | Promega | E1731 | |
Commercial assay, kit | DNA polymerase | Qiagen | 203203 | |
Commercial assay, kit | MinElute Kit | Qiagen | 28004 | |
Commercial assay, kit | T3 RNA polymerase | Promega | P2083 | |
Commercial assay, kit | DIG RNA labeling mix | Roche | 11277073910 | |
Commercial assay, kit | Micro Bio-Spin P-30 Gel Columns | Bio-Rad | 732–6223 | |
Commercial assay, kit | Tissue-Tek O.C.T. Compound | Sakura Finetek | 4583 | |
Commercial assay, kit | Superfrost Plus Slides | Fisherbrand | 1255015 | |
Commercial assay, kit | Parafilm | Sigma | P7793 | |
Commercial assay, kit | Blocking reagent | Roche | 11096176001 | |
Commercial assay, kit | Maleic acid | Sigma | M0375 | |
Antibody | Anti-DIG antibody (sheep polyclonal) | Roche | 11207733910 | 1:1000 |
Commercial assay, kit | TSA-Fluorescein | PerkinElmer | NEL741B001KT | 1:400 |
Antibody | pS6 Antibody for IF (rabbit polyclonal) | ThermoFisher | 44–923G | 1:300 |
Antibody | Anti-rabbit IgG Cy3 Antibody (donkey polyclonal) | Jackson Immuno | 711-165-152 | 1:200 |
Other | Bisbenzimide | Sigma | H 33258 | 1:100,000 |
Other | Paper bucket | International Paper | DFM85 | |
Other | Blotting pad | VWR | 28298–014 | |
Other | Odor cassette | Sakura Finetek | 0006772–01 | |
Commercial assay, kit | LoBind Tube | Eppendorf | 22431021 | |
Commercial assay, kit | NP40 Substitute | Sigma | 11332473001 | |
Commercial assay, kit | DHPC | Avanti Polar Lipids | 850306P | |
Antibody | pS6 Antibody for IP (rabbit monoclonal) | Cell Signaling | D68F8 | 6 μL |
Commercial assay, kit | Dynabeads Protein A | Invitrogen | 10002D | |
Commercial assay, kit | RNeasy Micro Kit | Qiagen | 74004 | |
Other | β-Caryophyllene | Sigma | W225207 | |
Other | 2,5-DMP | Sigma | 175420 | |
Other | 2-Heptanone | Sigma | W254401 | |
Other | (E)-β-Farnesene | Bedoukian | P3500-90 | |
Other | CPD | Sigma | C111201 | |
Other | MTMT | Lin et al., 2005 | Synthesized | |
Other | SBT | Meijer et al., 1973; Abrunhosa et al., 2001; Tashiro and Mori, 1999 | Synthesized | |
Other | DHB | Wiesler et al., 1984 | Synthesized |