Anti socs1 antibody

WT C57BL/6 mice aged from 6 to 8 weeks were obtained from the Experimental Animal Center of Chongqing Medical University. The lentiviral pLVX IRES Puro constructs to express GFP and PNPLA7-GFP Mission^® lentiviral pLKO.1 vectors encoding for scrambled shRNA or shRNAs targeting murine PNPLA7 were maintained in our lab [18 (link)]. LPS from Escherichia coli O111:B4 was obtained from Sigma. The MiniBEST Universal RNA Extraction Kit, PrimeScript™ II 1st Strand cDNA Synthesis Kit, and TB Green^® Fast qPCR Mix were purchased from TaKaRa. A PNPLA7 rabbit antiserum was generated in our lab by immunizing rabbits with a peptide in the regulatory domain of mouse PNPLA7. The generation of this antiserum and its specificity have been previously described [17 (link)]. The SIRT1 mouse monoclonal antibody (mAb) was purchased from Santa Cruz Biotechnology and β-actin mouse mAb, α-Tubulin mouse mAb, GAPDH mouse mAb, NF-κB p65 rabbit mAb, NF-κB1 p105/p50 rabbit mAb, iNOS antibody (Mouse Specific), IκBα (44D4) Rabbit mAb, p38 MAPK rabbit mAb, and Phospho-p38 MAPK (Thr180/Tyr182) rabbit mAb were obtained from Cell Signaling Technology. Anti-SOCS1 antibody and Anti-NF-κB p65 (acetyl K310) antibody were obtained from Abcam. Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor^® 647 conjugate was purchased from Thermo Fisher Scientific.

Following appropriate treatment, MC3T3-E1 cells were lysed using a protein extraction kit (Bio-Rad, Hercules, CA, USA). Equal quantities of protein samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were blocked in 5% fat-free milk for 2 h at room temperature before being probed with anti-AMPKα antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) (diluted at 1:1000 v:v), anti-p-AMPKα (Thr172) antibody (Cell Signaling Technology) (diluted at 1:1000 v:v), anti-SOCS1 antibody (Abcam, Cambridge, MA, USA) (diluted at 1:1000 v:v), or anti-β-actin antibody (Proteintech, Rosemont, IL, USA) (diluted at 1:2000 v:v) overnight at 4 °C. Protein bands were detected using an Odyssey CLx imaging system (LI-COR) followed by incubation for 2 h at ambient temperature with goat anti rabbit secondary antibody conjugated with DyLight fluorescent 800 (LI-COR) (diluted at 1:20,000 v:v).