Monoclonal rabbit

Rhodamine–phalloidin (for actin staining) was from Invitrogen (Carlsbad, CA); anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH), anti-HA, phospho-paxillin (Y118), monoclonal anti-Cdc42 antibodies, anti-GFP monoclonal antibody, active 9EG7 and total Hmβ1-1 CD29 β1-integrin, and monoclonal paxillin antibodies were from BD Biosciences (Palo Alto, CA); rat immunoglobulin G (IgG) a,κ and hamster IgG isotype controls were from Biolegend (San Diego, CA). Monoclonal rat from Roche Molecular Biochemicals (Indianapolis, IN) or monoclonal rabbit (from Cell Signaling Technology, Beverly, MA) anti-HA antibody was used for cell staining; antibodies against mouse FAK, phospho-FAK (Y397), vinculin, and α-tubulin were from Abcam (Cambridge, MA). Mouse monoclonal pan-arrestin F4C1 antibody recognizing epitope DGVVLVD in the N-domain was a generous gift of L. A. Donoso (Wills Eye Hospital, Philadelphia, PA). Arrestins were detected with arrestin-2-specific (1:6000; Mundell et al., 1999 (link)) or arrestin-3-specific (1:700; Orsini and Benovic, 1998 (link)) affinity-purified rabbit polyclonal antibodies. Total integrin antibody M-106 for Western blot was from Santa Cruz Biotechnology (Santa Cruz, CA).

All Western blots were run using a Mini-PROTEAN Tetra Cell system (Bio-rad, Hercules, CA). Both 10% and 4–20% gradient polyacrylamide gels were used. For gel electrophoresis, the running buffer was made up of 25 mM Tris, 192 mM glycine, and 0.1% (w/v) SDS at pH 8.3. Gels were run at constant voltage (200 v) for 30–50 min. For protein transfer to nitrocellulose membranes, transfer buffer was made up of 25 mM Tris, 0.2 M glycine, and 20% methanol at pH 8.5. A constant voltage (90 v) for 60 min was maintained for transfers. The transfer buffer was refrigerated prior to use, and the entire apparatus was kept cold (approx. 4 °C) during protein transfer. Following transfer, blots were incubated based on antibody manufacturer recommendations. For G6PD, GR, and β-actin quantification, blots were incubated for 12 h on a plate shaker at 4 °C with a polyclonal rabbit antibody (1:1000) (Cell Signaling, Danvers, MA) for G6PD, monoclonal mouse antibody (2 µg/mL) (Sigma-Aldrich, St. Louis, MO) for GR, and monoclonal rabbit (1:1000) (Cell Signaling, Danvers, MA) for β-actin. Both anti-rabbit and anti-mouse secondary antibodies (1:1000) were HRP-linked, and an HRP-detection kit followed by photo-development was used (Cell Signaling, Danvers, MA). All G6PD and GR bands were normalized to their corresponding β-actin bands and results are reported as % of β-actin.