P jnk1

HPASMCs serum starved in SMCM without growth factors for 48 h were treated for 2, 3 or 6 days with cocaine in presence or absence of Tat. Cells were lysed with RIPA buffer (Santa Cruz Biotechnology, Dallas, TX) followed by western blot for antibodies against TGFβRI, TGFβRII, BMPR-2, total and phosphorylated (p)-SMAD2/3, TAK1 (Santa Cruz Biotechnology, Dallas, TX), p-TAK1, p-MKK3/6, p-MKK4, p-p38MAPK, p-JNK1 (Cell signaling, Beverly, MA), p-TGFβRI (Abcam, Cambridge, MA), p-TGFβRII (Biorbyt, San Francisco, CA) and β-actin (Sigma-Aldrich, St. Louis, MO). For the detection of protein expression, enhanced chemiluminescence system (Thermo Scientific, Rockford, Illinois) was used after secondary probing with HRP-conjugated anti-mouse or anti-rabbit antibodies (Millipore, Billerica, MA). The density of bands was analyzed using the NIH ImageJ software. For co-immunoprecipitation total cellular protein (50 µg) was incubated at 4 °C, overnight with 1 µg of anti-p-TAK1 followed by immunoprecipitation using protein A/G agarose beads (Thermo Scientific) as per manufacturer’s instructions. Western blot was then performed to compare BMPR2-p-TAK1 complex with TGFβR2-p-TAK1 complex with 25 μg of precipitated protein using antibodies against BMPR2 and TGFβR2.