Qproteome cell fractionation kit

For total cell lysate (TCL) protein preparation, cells were disrupted in RIPA lysis buffer (150 mM NaCl, 300 mM sucrose, 1% Triton X-100, 0.5% sodium deoxycholate, 50 mM Tris-HCl, pH = 7.5) containing protease (Roche Diagnostics) and phosphatase inhibitors (Calbiochem-EMD) as previously described [14 (link)]. The HUVEC cellular fractions were isolated according to the protocol in the Qproteome cell fractionation kit (Qiagen). For co-immunoprecipitation studies, cell culture plates were washed three times with ice-cold PBS and lysates were prepared using a modified RIPA buffer containing protease and phosphatase inhibitors as above. Mouse lung tissue was disrupted using an Omni homogenizer for 5 seconds at low speed followed by using a QIAshredder homogenizer. Following normalization of protein concentrations by BCA assay, Western blot analysis was performed with α-zyxin (Synaptic Systems), α-endoglin (BD Biosciences), α-β-actin (Sigma-Aldrich), α-YAP (Santa Cruz sc-101199), α-14-3-3ζ (GeneTex), α-CYR61 (Santa Cruz sc-8561), α-CTGF (Santa Cruz sc-14939), α-Integrin α5 (Santa Cruz sc-10729), and α-Integrin β1 (BD Transduction Laboratories Inc.) antibodies.