Criterion 4 15 tris hcl gradient gels

Transfected 293T17 cells were treated with cell lysis buffer (Cell Signaling Technologies) with added complete mini protease inhibitor mixture tablets (Roche), phenylmethylsulfonyl fluoride and Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich). Lysates were spun at 8000 rpm for 10 min at 4 °C to pellet cell debris, mixed 2:1 with 3X GS sample buffer with β-ME (75 mM Tris (pH 6.8), 3% sodium dodecyl sulfate, 15% glycerol, 8% β-mercaptoethanol, and 0.1% bromphenol blue) and heated at 95 °C for 5 min. Lysates were run on Criterion 4–15% Tris-HCl gradient gels (Bio Rad), transferred to a PVDF membrane, and blocked for 30 min in Tris-buffered saline with Tween (TBST) with 5% BSA. Blots were probed overnight at 4 °C with anti-ACK1 (TNK2) rabbit antibody (Abcam, catalog no. ab65108), anti-ACK1 Phospho Y284 rabbit antibody (Abcam, ab74091), anti-ACK N-terminal antibody (Abcam ab137506), anti-actin mouse antibody (Millipore), followed by anti-rabbit or anti-mouse IgG HRP conjugate secondary antibodies. Blots were developed using Clarity chemiluminescent substrate (BioRad) or SuperSignal West Dura Extended Duration Substrate (Life Technologies) and imaged using a BioRad ChemiDoc MP Imaging System or exposed to x-ray film.

Cells were lysed in cell lysis buffer (Cell Signaling Technologies) containing complete mini protease inhibitor tablets (Roche). To pellet cellular debris the lysates were spun at 12000 rpm, 4ºC for 10 minutes and subsequently mixed with 3X SDS sample buffer (75mM Tris (pH 6.8), 3% SDS, 15% glycerol, 8% β-mercaptoethanol, and 0.1% bromophenol blue). Samples were incubated at 95ºC for 5 minutes and run on Criterion 4–15% Tris-HCl gradient gels (Bio Rad). Gels were transferred to PVDF membranes and blocked in Tris-buffered saline with Tween (TBST) with 5% Bovine Serum Albumin (BSA). Blots were probed with the following primary antibodies: anti-GCSF receptor Rabbit antibody (Abcam, cat# ab126167), anti-Flag Mouse antibody (Sigma, cat# F3165-1MG), anti-V5 Mouse antibody (Invitrogen, cat# 46-0705), and anti-Actin Mouse antibody (CalBioChem, cat# CP01). HRP conjugate secondary antibodies against mouse IgG and rabbit IgG (Promega) were used followed by imaging of the blots on a Lummi imager (Roche Applied Science). Co-immunoprecipitation experiments to measure dimerization were performed asdescribed previously (10 (link)).