Anti rbp4

Protein from tissues and cells was lysed by ice-cold RIPA lysis buffer, and the protein level was measured by the BCA Protein Assay Kit. After centrifugation at 12000 g at 4°C for 5 min, the same quantity of protein was extracted with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to polyvinylidene fluoride (PVDF) membranes. Then, the membranes were blocked in 5% skim milk. Afterwards, incubation was carried out with primary antibodies (1 : 1000, anti-RBP4 (Bioss, China); anti- Bcl-2 (Bioss, China) 1 : 300; anti-JAK2 (Bioss, China); 1 : 5000; anti-STAT3 (Bioss, China); 1 : 10000; anti-p-JAK2 (abcam, USA); 1 : 1000; anti-p-STAT3 (abcam, USA); 1 : 300, anti-Cyclin D1 (Bioss, China); 1 : 1000) at 4°C overnight. After washing with TBST for four times, the membranes were then cultured with secondary antibody horseradish peroxidase-conjugated goat anti-rabbit IgG (1 : 10000, Zs-BIO, China) for 1 h. Finally, the protein were visualized with an enhanced chemiluminescence (ECL) detection kit (Thermo, USA), and band intensity was quantified by densitometry with ImageJ software version 1.40 (National Institutes of Health, Bethesda, USA).