Hrp conjugate secondary antibodies against mouse igg and rabbit igg

Immunoblotting and co-IP were performed as described previously (3 (link)). Briefly, for reducing conditions, 8% β-mercaptoethanol was added into 3× SDS sample buffer [75 mmol/L Tris (pH 6.8), 3% SDS, 15% glycerol, and 0.1% bromophenol blue]. The mixed samples were boiled for 5 minutes in a 95°C heat block before loading on a 4% to 15% Tris-HCl gradient gel (Bio-Rad). For nonreducing conditions, no β-mercaptoethanol was added, and the nonboiled samples were loaded on a 10% gel. The following primary antibodies and reagents were used: anti-GCSF receptor antibody (Abcam, ab126167), anti-GFP antibody (Santa Cruz Biotechnology, sc-9996), anti-pSTAT3 Tyr705 (Cell Signaling Technology, 9131), anti-STAT3 (Cell Signaling Technology, 9132), anti-pSTAT5 Tyr694 (Cell Signaling Technology, 9151), anti-STAT5 (BD Transduction Laboratories, 610192), anti-pERK1/2 Thr202/Tyr204 (Cell Signaling Technology, 4370), anti-ERK1/2 (Cell Signaling Technology, 9102), anti-pJAK2 Tyr1007/1008 (Cell Signaling Technology, 3776), anti-JAK2 (Cell Signaling Technology, 3230), and HRP conjugate secondary antibodies against mouse IgG and rabbit IgG (Promega).

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)).