Rap1a rap1b 26b4 rabbit mab

8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (D-007) was purchased from Biolog Life Science Institute, Bremen, Germany. 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), Alamar blue cell viability reagent and SYPRO™ Orange protein stain were purchased from ThermoFisher Scientific, Waltham, MA, USA. Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) sodium salt, 0.33% neutral red solution, dimethyl sulfoxide (DMSO) for molecular biology and anti-Rabbit IgG, HRP conjugate, were obtained from Sigma-Aldrich, St. Louis, MO, USA. Rap1A/Rap1B (26B4) rabbit mAb, EPAC1 (DE3) mouse mAb and STAT3/phospho-STAT3 (Tyr 705) antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA.

After SDS-PAGE, samples were transferred onto a 0.45 μm polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). The membrane was blocked with 5% non-fat milk in TBST buffer overnight at 4 °C. After that, the membrane was incubated with primary antibody at RT for 3 h and then incubated with horeseradish peroxidase (HRP)-conjugated secondary antibody at RT for 1 h. The blots were detected by Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). The following primary antibodies were used in this study: 6xHis epitope tag antibody (Catalog# MA1-21315, Thermo Fisher Scientific), Rap1a/Rap1b (26B4) Rabbit mAb (Catalog# 2399, Cell Signaling Technology), Ras (27H5) Rabbit mAb (Catalog# 3339, Cell Signaling Technology), GAPDH (D16H11) Rabbit mAb (Catalog# 5174, Cell Signaling Technology), Anti-Talin-1 antibody (Catalog# ab57758, Abcam Inc.), and Anti-RIAM antibody (EPR2806; Catalog# ab92537, Abcam Inc.). The following secondary antibodies were used in this study: anti-mouse IgG, HRP-linked antibody (Catalog# 7076, Cell Signaling Technology), and anti-rabbit IgG, HRP-linked antibody (Catalog# 7074, Cell Signaling Technology). Primary antibodies were used at 1:1000 dilution, and secondary antibodies were used at 1:3000 dilution.