Anti rap1a

The total protein of cells and tissues was extracted using lysis buffer (Genechem, China) supplemented with protease inhibitor (Complete mini, USA). The protein concentration was measured using the bicinchonininc acid (BCA) protein assay kit (Beyotime Biotechnology, China).Then proteins were loaded and separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidenedifloride (PVDF) membranes. The membranes were subsequently blocked with 5% non-fat milk for 2 h and incubated with primary antibodies overnight at 4 °C. The primary antibodies used were as follows: anti-RAP1A (1:1000, Abcam, UK), anti-Forkhead box O3 (FOXO3) (1:1000, Cell Signaling Technology, USA), anti-PTEN (1:1000, Abcam), anti-cyclin D1 (CCND1, 1:1000, Abcam) and anti-β-actin (1:5000, Abcam) or anti-GAPDH (1:5000, Santa Cruz Biotechnology, USA). The membranes were incubated with secondary antibody (1:5000, Santa Cruz Biotechnology) for 1 h at room temperature. Finally, the blots were visualized using Chemiluminescence Detection Kit (Thermo Fisher Scientific). The protein expression was quantified using Quantity One software and β-actin or GAPDH served as the internal control.

The antibodies anti-FABP4 (number ab92501), anti-JAK1 (number ab47435), anti-JAK3 (number ab45141), anti-STAT1 (number ab30645), anti-STAT3 (number ab76315), anti-SOCS1 (number ab9870), anti-SOCS2 (number ab3692), and anti-Rap1a (number ab197673) were purchased from Abcam (Cambridge, MA). Anti-Src (number 2108S), anti-p-Src family (Tyr416 number 6943S), anti-STAT2 (number 72604S), anti-JAK2 (number 3230S), anti-p-JAK2 (Y1007/1008, number 3771S), and anti-p-STAT2 (number 88410S) antibodies were obtained from Cell Signaling Technology (Danvers, MA), while anti-β-actin (number sc-8432) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).