His epitope tag antibody

The dephosphorylation reaction was monitored by Western immunoblot analysis. For that, 1 µg of both His6‐Wzc and His6‐Wzb was incubated in 20 µl of reaction buffer (100 mM sodium citrate, pH 6.5, and 1 mM EDTA) at 30°C for 0, 1, 2, 4, 6, 12, or 24 hr. Reactions were terminated by adding SDS‐PAGE sample buffer. Samples were heated at 95°C for 5 min, separated on 4%–15% SDS‐PAGE gels (Bio‐Rad), and transferred onto nitrocellulose membranes as previously described (Leitao, Oxelfelt, Oliveira, Moradas‐Ferreira, & Tamagnini, 2005). Membranes were probed with either monoclonal anti‐phosphotyrosine antibody (PT‐66; Sigma) diluted 1:2,000, or 6x‐His Epitope Tag Antibody (Thermo Scientific) diluted 1:1,000. Membranes were then incubated with goat anti‐mouse IgG‐HRP (Santa Cruz Biotechnology) at a dilution of 1:5,000. Immunodetection was performed using the ECL^TM Western blotting detection reagents (GE healthcare) or the WesternBright^TM Quantum (Advansta) and a Chemi DocTM XRS+Imager (Bio‐Rad). The relative signal intensity of the bands obtained by immunodetection was quantified using the Image Lab software (Bio‐Rad). Data were statistically analyzed as described below.

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.