Rabbit anti gfp antibody

Subconfluent cells were washed with PBS, and lysed with RIPA lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris–HCl (pH 7.4), 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) with 1 mmol/L sodium orthovanadate, 1 mmol/L PMSF, and 1% cocktail of protease inhibitors (Sigma, St. Louis, MO). The lysates were clarified by centrifugation at 12000 g for 20 min at 4°C and separated by SDS-PAGE followed by transfer onto nitrocellulose membranes. The following antibodies were used: mouse anti-E-cadherin antibody (BD Biosciences, San Jose, CA), goat anti-biotin antibody (Sigma), mouse anti-GAPDH antibody (KangChen Bio-tech, Shanghai, China), rabbit anti-GFP antibody (Cell Signaling Technology, Beverly, MA), rabbit anti-Arf6 antibody (Abcam, Cambridge, MA). Protein bands were detected by incubating with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized with ECL reagent (Millipore). Digital images of immunoblots were obtained with a Chemidoc XRS and analyzed using the image analysis program Quantity One (Bio-Rad, Hercules, CA).

For preparation of yeast protein extracts, yeast cells with pYC2-CT vectors encoding BteA protein derivatives (Table S4) were induced for 20 h by cultivation in SC-galactose media. Equivalents of OD₆₀₀ = 1 of yeast cell cultures were collected, and denatured protein extracts were prepared by NaOH lysis/TCA precipitation method, according to (45 (link)). For preparation of mammalian cell protein extracts, HeLa cells at 40% confluency in a 6-well plate were transiently transfected with pEGFP-N2 vectors encoding BteA protein derivatives (Table S4) using Lipofectamine 2000 reagent (Invitrogen). Eighteen hours after transfection, cells were washed with PBS, lysed with 100 μl of ice-cold lysis buffer containing 0.2% Triton X-100 and complete mini protease inhibitors (EDTA free, Roche) in PBS, and clarified by centrifugation (5 min, 10,000g). Extracts were mixed with SDS-PAGE sample loading buffer, heated for 5 min at 50 °C, and separated on 10% SDS-PAGE gels. After the transfer onto nitrocellulose membrane, the proteins were probed overnight with rabbit anti-GFP antibody (1:2000; clone D5.1, Cell Signaling Technology) and revealed by horseradish peroxidase–conjugated anti-rabbit IgG secondary antibody (1:3000; GE Healthcare). Blots were developed using a Pierce ECL chemiluminescence substrate (Thermo Fisher Scientific) and an Image Quant LAS 4000 station (GE Healthcare).

Cells were solubilized for 30 min at 4 °C in lysis buffer containing 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, a protease inhibitor cocktail (0.3 μM aprotinin, 1 mM PMSF; Sigma-Aldrich and 10 μM leupeptin). After centrifugation at 12,000g for 20 min at 4 °C, the supernatants were transferred and the protein content was measured. Lysates (50 μg) were loaded on a 10% SDS–PAGE gel followed by transfer to Immobilon-P membrane (EMD Millipore/Merck, Damstadt, Germany). Membranes were blocked and probed with the appropriate antibodies. Anti-HA for IP (1 μg per assay) was obtained from Santa Cruz Biotechnology, and for Western blot (HA.11mAb; 1:500 dilution) from Covance, Berkeley, USA. 1 μg/assay SAM11 antibody (Santa Cruz Biotechnology) was used for IP and a 1:500 dilution was used for Western blot detection of overexpressed hPar2. Rabbit anti GFP antibody, anti Akt antibody and anti phospho-Akt were obtained from Cell Signaling Technology and used at a dilution of 1:1,000. Mouse anti T7 antibody was obtained from Novagen Madison, WI; and used at a dilution of 1:10,000. Anti β-actin was purchased from Sigma-Aldrich and used at a dilution of 1: 5,000. Uncropped immunbloots and larger blot areas are represented in Supplementary Fig. 12