Anti rock2 sc 398519

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.

Cells grown on coverslips were treated with verteporfin as described above. Cells were fixed in 4% paraformaldehyde were permeabilized with 0.15% Triton X-100 (Sigma) in phosphate-buffered saline or in methanol and were incubated with the following antibodies: anti-YAP (sc-271134, dilution 1:25), anti-β-catenin (sc-7963, dilution 1:50), and anti-ROCK2 (sc-398,519, dilution 1:50) that were all purchased from Santa Cruz Biotechnologies; and anti-N-cadherin (BD Transduction Labs, 610921, dilution 1:100). Anti-mouse FITC (Thermo Scientific, #31569, dilution 1:100) or anti-goat IgG NL493 (FITC equivalent R&D, #NL003, dilution 1:50) were used as secondary antibodies. Nuclei were counterstained with Hoechst 33256 (Sigma). Images were acquired using a Nikon ECLIPSE 90i microscope and were then analyzed with NIS-Elements software (Nikon).