Anti p300 sc 48343

Cytoplasmic and nuclear fractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Thermo scientific #78835) and subjected to western blot analysis or to co-IP. Co-IP specific antibodies (anti-ROCK1 (sc-6055; sc-374388) and anti-ROCK2 (sc-398519) from Santa Cruz Biotechnology; anti-ROCK2 (R8653) from Sigma; anti-pSTAT3 (Tyr705) (#4113) from Cell Signaling Technology) were immobilized on Dynalbeads ProteinG (Thermo Fisher Scientific Inc.) as instructed by manufacturer. JAK2 (D2E12) conjugated Sepharose beads were purchased from Cell Singling Technology (#4089). For co-IP, cytoplasmic or nuclear extracts were incubated overnight with the antibody-conjugated dynalbeads or sepharose beads. The beads were then washed with buffer (20 mM Tris-Hcl (pH 7.5), 20% glycerol, 0.1 mM EDTA, 300 mM NaCl, 0.5 mM DTT, 0.5 mM PMSF, 0.1% NP40) five times, and Tris/EDTA (TE) once. The bound proteins were eluted by incubating at 70 °C for 10 minutes in TE/1%SDS buffer and subjected to western blot analysis. Other antibodies used for western blot analysis are anti-phospho-MLC (Invitrogen PA5-17727); anti-P300 (SC-48343) and anti-JAK3 (SC-6932) from Santa Cruz Biotechnology; anti-MLC (#85053), anti-MYPT (#26345), anti-STAT5 (#94205) from Cell Signaling Technology.