Lactate transporter MCT1 in hepatic stellate cells promotes fibrotic collagen expression in nonalcoholic steatohepatitis
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, United States
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, United States
- Department of Neurology, Johns Hopkins School of Medicine, United States
Figures
Figure 1
MCT1 depletion attenuates transforming growth factor 1β (TGF-β1)-stimulated collagen 1 production in human LX2 stellate cells.
Cells were transfected with either NTC-siRNA or MCT1-siRNA for 6 hr. Then, cells were maintained in serum-starved media with or without 10 ng/ml of recombinant human TGF-β1 for 48 hr and harvested. …
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Figure 1—source data 1
MCT1 depletion attenuates transforming growth factor 1β (TGF-β1)-stimulated collagen 1 production in human LX2 stellate cells.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig1-data1-v1.zip
Figure 2
Screening of chemically modified Chol-MCT1-siRNA in vitro.
(A) Targeted regions of multiple Chol-MCT1-siRNA candidates on Slc16a1/Mct1 transcript. (B) Silencing efficacy of each Chol-MCT1-siRNA candidate (1.5 µM) on Slc16a1/Mct1 mRNA expression levels was …
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Figure 2—source data 1
Screening of chemically modified Chol-MCT1-siRNA in vitro.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig2-data1-v1.zip
Figure 3 with 1 supplement
Biodistribution of Chol- and GN-MCT1-siRNA in the liver.
Male C57BL/6 wild-type mice (16–18 weeks, n=4) were subcutaneously injected with 10 mg/kg of each siRNA, twice within 15 days, while fed a chow diet. Mice were sacrificed on day 15. (A) Chemical …
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Figure 3—source data 1
Biodistribution of Chol- and GN-MCT1-siRNA in the liver.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig3-data1-v1.zip
Figure 3—figure supplement 1
Biodistribution of GN-MCT1-siRNA and Chol-MCT1-siRNA.
Male C57BL/6 wild-type mice (16–18 weeks, n=4) were subcutaneously injected with 10 mg/kg of siRNAs twice within 15 days. Livers were perfused through inferior vena cava and multiple liver cells …
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Figure 3—figure supplement 1—source data 1
Biodistribution of GN-MCT1-siRNA and Chol-MCT1-siRNA.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig3-figsupp1-data1-v1.zip
Figure 4 with 2 supplements
Hepatic MCT1 depletion did not resolve steatosis in a genetically obese nonalcoholic steatohepatitis (NASH) mouse model.
(A) Male ob/ob mice (10 weeks, n=6) were subcutaneously injected with 10 mg/kg of siRNA once every 10 days. Mice were fed a Gubra Amylin NASH (GAN) diet for 3 weeks and sacrificed. (B) Livers were …
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Figure 4—source code 1
Hepatic MCT1 depletion did not resolve steatosis in a genetically obese nonalcoholic steatohepatitis (NASH) mouse model.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig4-code1-v1.zip
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Figure 4—source data 1
Hepatic MCT1 depletion did not resolve steatosis in a genetically obese nonalcoholic steatohepatitis (NASH) mouse model.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig4-data1-v1.zip
Figure 4—figure supplement 1
GN-MCT1-siRNA induced a complementary effect on monocarboxylate transporter (MCT) isoform expression and decreased food intake and body weight.
Male ob/ob mice (10 weeks, n=6) were subcutaneously injected with 10 mg/kg of siRNA once every 10 days. Mice were fed a Gubra Amylin NASH (GAN) diet for 3 weeks. (A, B) The complementary effect of …
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Figure 4—figure supplement 1—source data 1
GN-MCT1-siRNA induced a complementary effect on monocarboxylate transporter (MCT) isoform expression and decreased food intake and body weight.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig4-figsupp1-data1-v1.zip
Figure 4—figure supplement 2
Both Chol-MCT1-siRNA and GN-MCT1-siRNA significantly decreased hepatic DNL gene expression.
Male ob/ob mice (10weeks, n=6) were subcutaneously injected with 10mg/kg of siRNA once every 10days. Mice were fed a Gubra Amylin NASH (GAN) diet for 3weeks and sacrificed. Representative DNL gene …
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Figure 4—figure supplement 2—source data 1
Both Chol-MCT1-siRNA and GN-MCT1-siRNA significantly decreased hepatic DNL gene expression.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig4-figsupp2-data1-v1.zip
Figure 5 with 2 supplements
Opposite effects of Chol-MCT1-siRNA versus GN-MCT1-siRNA on fibrotic type 1 collagen expression.
Male ob/ob mice (10weeks, n=6) were subcutaneously injected with 10mg/kg of siRNA once every 10days. Mice were fed a Gubra Amylin NASH (GAN) diet for 3weeks and sacrificed. Representative fibrogenic …
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Figure 5—source code 1
Opposite effects of Chol-MCT1-siRNA versus GN-MCT1-siRNA on fibrotic type 1 collagen expression.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig5-code1-v1.zip
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Figure 5—source data 1
Opposite effects of Chol-MCT1-siRNA versus GN-MCT1-siRNA on fibrotic type 1 collagen expression.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig5-data1-v1.zip
Figure 5—figure supplement 1
A comparable level of M1/M2 macrophage polarization upon Chol-MCT1-siRNA and GN-MCT1-siRNA administration.
Male ob/ob mice (10weeks, n=6) were subcutaneously injected with 10mg/kg of siRNA once every 10days. Mice were fed a Gubra Amylin NASH (GAN) diet for 3weeks and sacrificed. Representative …
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Figure 5—figure supplement 1—source data 1
A comparable level of M1/M2 macrophage polarization upon Chol-MCT1-siRNA and GN-MCT1-siRNA administration.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig5-figsupp1-data1-v1.zip
Figure 5—figure supplement 2
Intravenous injection of AAV9-Lrat-Cre in MCT1fl/fl mice specifically targets hepatic stellate cells.
(A) Male mice (9–10 weeks, n=4) were intravenously injected with 1×1011 gc of either AAV9-Lrat-null or AAV9-Lrat-Cre. Mice were fed a chow diet for 3 weeks and sacrificed. Isolation of either (B) …
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Figure 5—figure supplement 2—source data 1
Intravenous injection of AAV9-Lrat-Cre in MCT1fl/fl mice specifically targets hepatic stellate cells.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig5-figsupp2-data1-v1.zip
Figure 6
MCT1 depletion did not resolve steatosis in the choline-deficient, high-fat diet (CDHFD)-induced nonalcoholic steatohepatitis (NASH) model.
(A) Male MCT1fl/fl mice (8 weeks, n=10) were intravenously injected with 2×1011 gc of AAV-TBG-Cre or AAV-Lrat-Cre or both. The same amount of AAV-TBG-null or AAV-Lrat-null was used as a control. A …
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Figure 6—source code 1
MCT1 depletion did not resolve steatosis in the choline-deficient, high-fat diet (CDHFD)-induced nonalcoholic steatohepatitis (NASH) model.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig6-code1-v1.zip
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Figure 6—source data 1
MCT1 depletion did not resolve steatosis in the choline-deficient, high-fat diet (CDHFD)-induced nonalcoholic steatohepatitis (NASH) model.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig6-data1-v1.zip
Figure 7
Hepatocyte-specific MCT1KO accelerated fibrosis, while hepatic stellate cell-specific MCT1KO decreased it.
Male MCT1fl/fl mice (6 weeks, n=10) were intravenously injected with 2×1011 gc of AAV-TBG-Cre or AAV-Lrat-Cre or both. The same amount of AAV-TBG-null or AAV-Lrat-null was used as a control. A week …
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Figure 7—source code 1
Hepatocyte-specific MCT1KO accelerated fibrosis, while hepatic stellate cell-specific MCT1KO decreased it.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig7-code1-v1.zip
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Figure 7—source data 1
Hepatocyte-specific MCT1KO accelerated fibrosis, while hepatic stellate cell-specific MCT1KO decreased it.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig7-data1-v1.zip
Figure 8 with 3 supplements
Lactate enhances the transforming growth factor 1β (TGF-β1)-stimulatory effect in the presence of pyruvate in human LX2 stellate cells.
Cells were treated with increasing doses of sodium lactate (0, 2.5, 5, 10, 20, 40 mM) for 48 hr. Dose-response effect of sodium lactate on (A) COL1A1 mRNA and (B) collagen 1 protein levels were …
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Figure 8—source data 1
Lactate enhances the transforming growth factor 1β (TGF-β1)-stimulatory effect in the presence of pyruvate in human LX2 stellate cells.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig8-data1-v1.zip
Figure 8—figure supplement 1
MCT1 promotes SMAD3 phosphorylation/activation in human LX2 stellate cells.
Cells were transfected with either NTC-siRNA or MCT1-siRNA for 6 hr. Then, cells were maintained in serum-starved media with or without 10 ng/ml of recombinant human transforming growth factor 1β …
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Figure 8—figure supplement 1—source data 1
MCT1 promotes SMAD3 phosphorylation/activation in human LX2 stellate cells.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig8-figsupp1-data1-v1.zip
Figure 8—figure supplement 2
MCT1 silencing enhanced fibrogenic gene expression levels in human hematoma cell lines, HepG2.
Cells were transfected with either NTC-siRNA or MCT1-siRNA for 6 hr. 48 hr later, cells were harvested, and media were collected. LX2 cells were provided with the conditioned media (40% conditioned …
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Figure 8—figure supplement 2—source data 1
MCT1 silencing enhanced fibrogenic gene expression levels in human hematoma cell lines, HepG2.
- https://cdn.elifesciences.org/articles/89136/elife-89136-fig8-figsupp2-data1-v1.zip
Figure 8—figure supplement 3
Graphical abstract.
Hepatocyte MCT1KO enhances fibrosis, while stellate cell MCT1KO decreases it.
© 2024, BioRender Inc. This figure was created with BioRender.com, and published under a CC-BY-NC-ND license with permission.
Tables
Table 1
Sequences of chemically modified siRNA candidates targeting MCT1 used in in vitro screening.
siRNAs utilized in in vitro screening were a double-strand oligonucleotide comprised of 15 sense and 20 antisense nucleotides. The sequences of each candidate’s antisense and sense strands were …
| Antisense strands: | |
|---|---|
| Oligo ID | Chemically modified RNA sequence |
| MCT1-507 | P(mU)#(fG)#(mU)(fU)(mA)(fC)(mA)(fG)(mA)(fA)(mA)(fG)(mA)#(fA)#(mG)#(fC)#(mU)#(fG)#(mC)#(fG) |
| MCT1-1976 | P(mU)#(fA)#(mA)(fA)(mC)(fU)(mU)(fA)(mA)(fG)(mG)(fC)(mA)#(fC)#(mA)#(fU)#(mA)#(fU)#(mU)#(fA) |
| MCT1-2013 | P(mU)#(fU)#(mU)(fA)(mA)(fA)(mA)(fG)(mU)(fU)(mA)(fA)(mG)#(fG)#(mC)#(fU)#(mC)#(fU)#(mC)#(fU) |
| MCT1-2042 | P(mU)#(fU)#(mU)(fA)(mA)(fA)(mA)(fC)(mA)(fA)(mA)(fU)(mG)#(fA)#(mA)#(fU)#(mU)#(fC)#(mA)#(fG) |
| MCT1-2060 | P(mU)#(fU)#(mU)(fC)(mC)(fU)(mU)(fU)(mU)(fA)(mA)(fA)(mA)#(fU)#(mG)#(fA)#(mC)#(fA)#(mU)#(fU) |
| MCT1-2120 | P(mU)#(fU)#(mU)(fA)(mC)(fA)(mA)(fA)(mC)(fA)(mA)(fC)(mA)#(fA)#(mC)#(fA)#(mA)#(fA)#(mA)#(fC) |
| MCT1-3067 | P(mU)#(fU)#(mU)(fU)(mC)(fU)(mG)(fC)(mC)(fU)(mC)(fU)(mA)#(fU)#(mU)#(fC)#(mA)#(fG)#(mA)#(fA) |
| MCT1-3160 | P(mU)#(fU)#(mC)(fU)(mU)(fA)(mC)(fA)(mC)(fA)(mA)(fG)(mG)#(fU)#(mU)#(fU)#(mU)#(fA)#(mA)#(fA) |
| MCT1-3290 | P(mU)#(fA)#(mU)(fA)(mU)(fU)(mA)(fG)(mA)(fA)(mA)(fG)(mG)#(fU)#(mU)#(fA)#(mA)#(fA)#(mA)#(fU) |
| MCT1-4340 | P(mU)#(fU)#(mG)(fA)(mA)(fU)(mU)(fU)(mG)(fU)(mA)(fU)(mG)#(fA)#(mG)#(fA)#(mA)#(fU)#(mA)#(fA) |
| Sense strands: | |
| Oligo ID | Chemically modified RNA sequence |
| Chol-MCT1-507 | (fC)#(mU)#(fU)(mC)(fU)(mU)(fU)(mC)(fU)(mG)(fU)(mA)(fA)#(mC)#(fA)-Chol |
| Chol-MCT1-1976 | (fU)#(mG)#(fU)(mG)(fC)(mC)(fU)(mU)(fA)(mA)(fG)(mU)(fU)#(mU)#(fA)-Chol |
| Chol-MCT1-2013 | (fG)#(mC)#(fC)(mU)(fU)(mA)(fA)(mC)(fU)(mU)(fU)(mU)(fA)#(mA)#(fA)-Chol |
| Chol-MCT1-2042 | (fU)#(mU)#(fC)(mA)(fU)(mU)(fU)(mG)(fU)(mU)(fU)(mU)(fA)#(mA)#(fA)-Chol |
| Chol-MCT1-2060 | (fC)#(mA)#(fU)(mU)(fU)(mU)(fA)(mA)(fA)(mA)(fG)(mG)(fA)#(mA)#(fA)-Chol |
| Chol-MCT1-2120 | (fG)#(mU)#(fU)(mG)(fU)(mU)(fG)(mU)(fU)(mU)(fG)(mU)(fA)#(mA)#(fA)-Chol |
| Chol-MCT1-3067 | (fA)#(mA)#(fU)(mA)(fG)(mA)(fG)(mG)(fC)(mA)(fG)(mA)(fA)#(mA)#(fA)-Chol |
| Chol-MCT1-3160 | (fA)#(mA)#(fC)(mC)(fU)(mU)(fG)(mU)(fG)(mU)(fA)(mA)(fG)#(mA)#(fA)-Chol |
| Chol-MCT1-3290 | (fA)#(mA)#(fC)(mC)(fU)(mU)(fU)(mC)(fU)(mA)(fA)(mU)(fA)#(mU)#(fA)-Chol |
| Chol-MCT1-4340 | (fC)#(mU)#(fC)(mA)(fU)(mA)(fC)(mA)(fA)(mA)(fU)(mU)(fC)#(mA)#(fA)-Chol |
Table 2
Sequences of the selected final chemically modified siRNA candidates targeting MCT1 used for in vivo studies.
MCT1-3160 was selected for the final construct for in vivo studies. MCT1-siRNAs utilized in in vivo study was a double-strand oligonucleotide comprised of 18 sense and 20 antisense nucleotides. To …
| Antisense strands: | |
|---|---|
| Oligo ID | Chemically modified RNA sequence |
| MCT1-3160 | VP(mU)#(fU)#(mC)(mU)(mU)(fA)(mC)(mA)(mC)(mA)(mA)(mG)(mG)#(fU)#(mU)#(fU)#(mU)#(mA)#(mA)#(fA) |
| Sense strands: | |
| Oligo ID | Chemically modified RNA sequence |
| Chol-MCT1-3160 | (mU)#(mA)#(mA)(mA)(mA)(mC)(mC)(fU)(fU)(fG)(mU)(fG)(mU)(mA)(mA)(mG)#(mA)#(mA)-Chol |
| GN-MCT-3160 | (mU)#(mA)#(mA)(mA)(mA)(mC)(mC)(fU)(fU)(fG)(mU)(fG)(mU)(mA)(mA)(mG)#(mA)#(mA)-GN |
Table 3
List of primers used for real-time quantitative PCR (rt-qPCR).
| Mouse primers: | ||
|---|---|---|
| Gene | Forward | Reverse |
| Slc16a1/Mct1 | TGTTAGTCGGAGCCTTCATTTC | CACTGGTCGTTGCACTGAATA |
| Slc16a1Mct1 (Exon 2,3 overlapping) | TGCAACGACCAGTGAAGTATC | GCTGCCGTATTTATTCACCAAG |
| Slc16a7/Mct2 | CCATCAGTAGTGTGTTGGTGAA | TCTATCACGCTGTTGCTGTAAG |
| Slc16a3/Mct4 | AGTGCCATTGGTCTCGTG | CATACTTGTAAACTTTGGTTGCATC |
| Srebf1 | GGAGCCATGGATTGCACATT | GGCCCGGGAAGTCACTGT |
| Mlxipl | TCTGCAGATCGCGTGGAG | CTTGTCCCGGCATAGCAAC |
| Fasn | GGAGGTGGTGATAGCCGGTAT | TGGGTAATCCATAGAGCCCAG |
| Scd1 | CCGGAGACCCCTTAGATCGA | TAGCCTGTAAAAGATTTCTGCAAACC |
| Acly | TGGTGGAATGCTGGACAA | GCCCTCATAGACACCATCTG |
| Tgfb1 | CTCCCGTGGCTTCTAGTGC | GCCTTAGTTTGGACAGGATCTG |
| Ihh | CTCTTGCCTACAAGCAGTTCA | CCGTGTTCTCCTCGTCCTT |
| Acta2 | ATGCTCCCAGGGCTGTTTTCC | GTGGTGCCAGATCTTTTCCATGTCG |
| Gli2 | CAACGCCTACTCTCCCAGAC | GAGCCTTGATGTACTGTACCAC |
| Gli3 | CACAGCTCTACGGCGACTG | CTGCATAGTGATTGCGTTTCTTC |
| Col1a1 | GCTCCTCTTAGGGGCCACT | CCACGTCTCACCATTGGGG |
| Col1a2 | GTAACTTCGTGCCTAGCAACA | CCTTTGTCAGAATACTGAGCAGC |
| Col3a1 | CTGTAACATGGAAACTGGGGAAA | CCATAGCTGAACTGAAAACCACC |
| Timp1 | CTCAAAGACCTATAGTGCTGGC | CAAAGTGACGGCTCTGGTAG |
| Alb | TGCTTTTTCCAGGGGTGTGTT | TTACTTCCTGCACTAATTTGGCA |
| Des | CTAAAGGATGAGATGGCCCG | GAAGGTCTGGATAGGAAGGTTG |
| Clec4f | GAGGCCGAGCTGAACAGAG | TGTGAAGCCACCACAAAAAGAG |
| B2m | CATGGCTCGCTCGGTGAC | CAGTTCAGTATGTTCGGCTTCC |
| F4/80 | CTTTGGCTATGGGCTTCCAGTC | GCAAGGAGGACAGAGTTTATCGTG |
| Ccl2 | AGGTCCCTGTCATGCTTCTG | AAGGCATCACAGTCCGAGTC |
| Il1b | TTTGACAGTGATGAGAATGACC | CTCTTGTTGATGTGCTGCTG |
| Tlr4 | ATGGCATGGCTTACACCACC | GAGGCCAATTTTGTCTCCACA |
| Ccr2 | ATCCACGGCATACTATCAACATC | CAAGGCTCACCATCATCGTAG |
| Ccl20 | GCCTCTCGTACATACAGACGC | CCAGTTCTGCTTTGGATCAGC |
| Cd163 | ATGGGTGGACACAGAATGGTT | CAGGAGCGTTAGTGACAGCAG |
| Arg1 | CTCCAAGCCAAAGTCCTTAGAG | AGGAGCTGTCATTAGGGACATC |
| Ccl22 | AGGTCCCTATGGTGCCAATGT | CGGCAGGATTTTGAGGTCCA |
| Cd206 | CTCTGTTCAGCTATTGGACGC | TGGCACTCCCAAACATAATTTGA |
| Human primers: | ||
| Gene | Forward | Reverse |
| SLC16A1/MCT1 | TGGAAGACACCCTAAACAAGAG | AAAGCCTCTGTGGGTGAATAG |
| ACTA2 | AGCGTGGCTATTCCTTCGT | CTCATTTTCAAAGTCCAGAGCTACA |
| TGFB1 | CAACGAAATCTATGACAAGTTCAAGCAG | CTTCTCGGAGCTCTGATGTG |
| COL1A1 | ACGTCCTGGTGAAGTTGGTC | ACCAGGGAAGCCTCTCTCTC |
| TIMP1 | AATTCCGACCTCGTCATCAGG | ATCCCCTAAGGCTTGGAACC |
| ACTB | GATGAGATTGGCATGGCTTT | GAGAAGTGGGGTGGCTT |
Table 4
List of antibodies used in this study.
| Reagent | Source | Identifier |
|---|---|---|
| Anti-MCT1 | Proteintech | Cat # 20139-1-AP |
| Anti-FASN | Cell Signaling | Cat # 3180s |
| Anti-ACLY | Cell Signaling | Cat # 4332 |
| Anti-SCD1 | Cell Signaling | Cat # 2794s |
| Anti-ChREBP | Novus Bio | Cat # NB400-135 |
| Anti-SREBP1 | Millipore | Cat # MABS1987 |
| Anti-GAPDH-HRP | Cell Signaling | Cat # 8884s |
| Anti-H3 | Cell Signaling | Cat # 4499s |
| Anti-Tubulin | Sigma-Aldrich | Cat # T5168 |
| Anti-pAMPK (T172) | Cell Signaling | Cat # 2535s |
| Anti-AMPKα | Cell Signaling | Cat # 2793s |
| Anti-αSMA | Cell Signaling | Cat # 19,245s |
| Anti-Collagen 1 | Southern Biotech | Cat # 1310–01 |
| Anti-HSP90-HRP | Cell Signaling | Cat # 79,631s |
| Anti-SMAD3 | Cell Signaling | Cat # 9523S |
| Anti-pSMAD3 | Abcam | Cat # AB52903 |
| Goat Anti-Rabbit IgG-HRP | Invitrogen | Cat # 65-6120 |
| Goat Anti-Mouse IgG-HRP | Invitrogen | Cat # 65-6520 |
| Goat Anti-Mouse IgG-HRP | Thermo Fisher | Cat # G21040 |
| Mouse Anti-Goat IgG-HRP | Santa Cruz | Cat # sc-2354 |
| Goat-anti-Rabbit-488 | Thermo Fisher | Cat # A11008 |
| ProLong Gold Antifade Mountant | Thermo Fisher | Cat # P36931 |
