(A) Structure of fusion protein TLR2-4. Total RNA was extracted from goat peripheral blood and was reverse- transcribed into cDNA. TLR2 extracellular domain and TLR4 transmembrane and intracellular …
The original blots of Toll-like receptor 2-4 (TLR2-4) (TLR2-4 blot was repeated twice) and wild-type (WT) fibroblasts.
The original blots.
Establishment of the TLR2-4 fibroblast cell line. (A) Gene editing efficiency was evaluated by the T7E1 cleavage assay. Fibroblasts were individually transfected with the eight small guide RNA …
The original gels of Figure 1—figure supplement 1A.
The original gels of Figure 1—figure supplement 1C.
The original gels of Figure 1—figure supplement 1C.
The effect of TLR2-4 on global transcriptome in killed S. aureus-infected fibroblasts. Both TLR2-4 and WT fibroblasts with knockdown endogenous TLR2 were stimulated with heat-killed S. aureus for 10 …
(A) The clone goat expressing TLR2-4 was prepared by somatic cell nuclear transfer. (B) Schematic representation of the site-specific integration of primers S-M and T-R. (C) Genomic DNA of …
The original gels of Figure 2C.
The original gels of Figure 2E.
The original gels of Figure 2F.
The original blots of Figure 2G.
And the ligand recognition function of TLR2-4 receptor in macrophages was identified. The effect of TLR2-4 on the phagocytose of S. aureus in macrophages. (A) The PCR products of 3795 bp were …
The original blots of Figure 2—figure supplement 1H.
(A) S. aureus burden in macrophages was estimated by the mean fluorescence intensity (MFI) of macrophages infected with fluorescein isothiocyanate (FITC)-labeled S. aureus using flow cytometry …
The original blots of Figure 3F.
The original blots of Figure 3G.
Macrophages from the Toll-like receptor 2-4 (TLR2-4) and two wild-type goats (WT1, WT2) were infected with S. aureus for 1 hr (multiplicity of infection [MOI] = 10), and then after washing three …
RNA sequencing of S. aureus infected TLR2-4 and WT macrophages. Macrophages were infected with S. aureus for 1 hr (multiplicity of infection [MOI] = 10). Cells were washed three times using PBS, and …
(A) The change in phosphorylation of JNK, ERK, and p38 in S. aureus-infected macrophages. Macrophages were pretreated with DMSO (carrier), Takinib (200 μM), or Amlexanox (100 μM) for 12 hr, then …
The original blots of Figure 5A, B and D.
The original blots of Figure 5A,B,D.
The original blots of Figure 5A,B,D.
Signaling pathways involved in S. aureus-infected TLR2-4 macrophages. (A) The protein level of MyD88 in macrophages was detected by Western blot. Tubulin was used to normalize the data of each …
The original blots of Figure 5—figure supplement 1A.
The original blots of Figure 5—figure supplement 1B.
The original blots of Figure 5—figure supplement 1D.
(A) TLR2-4 promoted the expression of ATG5 and ATG12 in macrophages after S. aureus infection. The mRNA relative expression of ATG5 and ATG12 was monitored by real-time reverse transcription PCR …
The original blots of Figure 6D, E and G.
The original blots of Figure 6D,E,G.
The original blots of Figure 6D,E,G.
Upon S. aureus stimulation, TLR2-4 on the surface of macrophages augmented LC3-promoted phagophore formation to enhance xenophagy through the MyD88-dependent TAK1-JNK/ERK signaling. Internalized …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Capra hircus) | TLR2 | GenBank | NC_030824.1 | |
Gene (Capra hircus) | TLR4 | GenBank | NC_030815.1 | |
Strain, strain background (Staphylococcus aureus) | ATCC29213 | GenBank | U77328 | Bacteria cells |
Transfected construct | PX458 (pSpCas9(BB)–2A-GFP) | Addgene | RRID: Addgene_48138 | PMID:24157548 |
Transfected construct | pRosa26-promoter | Addgene | RRID: Addgene_21710 | PMID:9108056 |
Transfected construct (Capra hircus) | siRNA to endogenous TLR2 | GenePharma | Sense: 5’-GCACUUCAACCCUCCCUUUTT-3’ | Antisense: 5’-AAAGGGAGGGUUGAAGUGCTT-3’ |
Sequence-based reagent | siRNA: nontargetin control | GenePharma | Silencer Select | |
Antibody | nti-MyC (Mouse monoclonal) | Proteintech | Cat# 60003–2-lg RRID: AB_2883088 | IF (1:100), WB (1:1000) |
Antibody | Anti-NF-kB p65 antibody (Rabbit polyclonal) | Abcam | Cat# ab16502 RRID: AB_443394 | WB (1:1000) |
Antibody | Anti-LMNB2 (Rabbit polyclonal) | Beyotime | Cat#: AF0219 | WB (1:1000) |
Antibody | Phospho-p44/42 MAPK (Rabbit polyclonal) | CST | Cat#: 9,101 RRID: AB_331646 | WB (1:1000) |
Antibody | Phospho-JNK1/2/3-T183/T183/T221 (Rabbit monoclonal) | ABclonal | Cat#: AP0631 RRID: AB_2771232 | WB (1:1000) |
Antibody | Tubulin (Mouse monoclonal) | Beyotime | Cat#: AT819 | WB (1:1000) |
Antibody | Anti-LC3 (Rabbit polyclonal) | Proteintech | Cat#: 14600–1-AP RRID: AB_2137737 | WB (1:1000) |
Antibody | Anti-SQSTM1/p62 (Rabbit polyclonal) | Affinity | Cat#: AF5384 RRID: AB_2837869 | WB (1:1000) |
Antibody | Phospho-p38 MAPK-T180 (Rabbit polyclonal) | ABclonal | Cat#: AP0238 RRID: AB_2771307 | WB (1:1000) |
Antibody | Anti-GAPDH (Rabbit polyclonal) | Sangon Biotech | Cat#: D110016 RRID: AB_2904600 | WB (1:1000) |
Antibody | Anti-TFEB (Rabbit polyclonal) | Proteintech | Cat#: 13372–1-AP RRID: AB_2199611 | WB (1:1000) |
Antibody | Anti-Optineurin (Rabbit polyclonal) | Affinity | Cat#: DF6655 RRID: AB_2838617 | WB (1:1000) |
Antibody | Phospho-pan-AKT1/2/3 (Rabbit polyclonal) | Affinity | Cat#: AF0908 RRID: AB_2834079 | WB (1:1000) |
Antibody | Phospho-FOXO1A (Rabbit polyclonal) | Affinity | Cat#: AF3416 RRID: AB_2834858 | WB (1:1000) |
Antibody | Acetyl-FOXO1A (Rabbit polyclonal) | Affinity | Cat#: AF2305 RRID: AB_2845319 | WB (1:1000) |
Antibody | Anti-PRKACA (Rabbit polyclonal) | ABclonal | Cat#: A0798 RRID: AB_2757400 | WB (1:1000) |
Antibody | Phospho-IKB alpha (Rabbit monoclonal) | CST | Cat#: 2,859 RRID: AB_561111 | WB (1:1000) |
Antibody | Phospho-IRF3-S386 (Rabbit polyclonal) | ABclonal | Cat#: AP0857 RRID: AB_2771209 | WB (1:1000) |
Antibody | Peroxidase-Conjugated Goat anti-Rabbit IgG | ZSGB-BIO | Cat#: ZB-2301 RRID: AB_2747412 | WB (1:5000) |
Antibody | Peroxidase-Conjugated Goat anti-Mouse IgG | ZSGB-BIO | Cat#: ZB-2305 RRID: AB_2747415 | WB (1:5000) |
Antibody | Alexa Fluor 594 goat anti-mouse IgG | ThermoFisher Scientific | Cat#: A11005 RRID: AB_2534073 | IF (1 μg/ml) |
Commercial assay or kit | Nuclear Extraction Kits for Cells | Invent Biotechnologies | Cat#: SC-003 | |
Chemical compound, drug | Dynasore | MCE | Cat#: HY-15304 | 50 μM |
Chemical compound, drug | SP600125 | MCE | Cat#: HY-12041 | 200 μM |
Chemical compound, drug | PD98059 | MCE | Cat#: HY-12028 | 200 μM |
Chemical compound, drug | Takinib | MCE | Cat#: HY-103490 | 200 μM |
Chemical compound, drug | Amlexanox | MCE | Cat#: HY-B0713 | 100 μM |
Software | ImageJ | http://imagej.nih.gov/ij/ | RRID: SCR_003070 | |
Software | Flowjo | https://www.flowjo.com/solutions/flowjo | RRID: SCR_008520 | |
Software | Origin | http://www.originlab.com/index.aspx?go=PRODUCTS/Origin | RRID: SCR_014212 | |
Other | DAPI stain | Solarbio | Cat#: C0065 | 10 μg/ml |
Quantification and statistical analysis.
The data for generation of clone goat and part primers used in this study.
(A) Table displaying the generation of clone goats by nuclear transfer. (B) Primers of crRNA-oligo. (C) Table displaying the sequences of primers in T7 endonuclease 1 (T7E1) assay. (D) All primers used for PCR. (E) Table displaying the primers for real-time reverse transcription PCR (qRT-PCR).