Anti tubulin antibody

BmN cells transfected with the recombinant plasmids, pSL1180-Myc-Masc, pSL1180-Myc-Masc-S and pSL1180-Myc-MascR, respectively, were lysed in western and immunoprecipitation buffer (pH 7.5) containing 20 mmol/L Tris–HCl, 150 mmol/L NaCl, and 1% Triton X-100 (Beyotime, Shanghai, China). The lysates were mixed with loading buffer and boiled for 5 min. Proteins were separated by 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and electroblotted onto polyvinylidene fluoride (PVDF) membranes (Roche). The membranes were blocked overnight at 4 °C in 5% (w/v) skimmed milk, and directly incubated with the anti-Myc-tag antibody for 2 h at 37 °C and washed three times. Finally, the membranes were incubated with SuperSignal (R) West Pico chemiluminescent substrate (Thermo Fisher Scientific) and photographed using a ChemiScope chemiluminescent imaging system. The anti-tubulin antibody (Beyotime) were used to detect tubulin that was used as an internal control.

Lipofectamine 2000, G418 and Opti-MEM were purchased from Invitrogen. Cell culture media and fetal bovine serum were obtained from Hyclone. Pertussis toxin (PTX), Go6983 and thapsigargin were purchased from Sigma. UBO-QIC was purchased from Dr. E. Kostenis at the University of Bonn. U0126, tyrphostin AG1478, GM6001, PP2 and ET-18-och3 were from Calbiochem. Monoclonal anti-FLAG antibody was purchased from BD Biosciences Pharmingen. Anti-phospho-ERK1/2 (Thr-202/Tyr-204) and ERK1/2 antibodies and horseradish peroxidase-conjugated anti-rabbit IgG were from Cell Signaling Technology. Anti-tubulin antibody was from Beyotime. The β-adrenergic receptor kinase COOH domain (495-689aa), adrenergic and the Gα subunit of transducin plasmid were from the lab of Dr. Naiming Zhou in Zhejiang University.