Monoclonal gst antibody

We followed essentially the procedures established by Cochrane et al.^{22 (link)} with minor modifications. Briefly, NMVMs were lysed in RIPA lysis buffer (50 mM Tris/pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, leupeptin, and protease inhibitors) supplemented with protease inhibitors and the lysate was immunoprecipitated with a monoclonal Flag antibody (Sigma, USA) at 4 °C. As a negative control, an equal amount of the lysate was immunoprecipitated with a monoclonal GST antibody (Santa Cruz Biotechnology, CA, USA). The immune complexes were collected with protein A/G plus-agarose beads (Santa Cruz Biotechnology, CA) and washed ten times with PBS. Flag-CIP85 and Cx43 were detected by immunoblotting. The physical interaction between endogenous CIP85 and Cx43 was explored by precipitating with the CIP85 antibody or a polyclonal Flag antibody (Santa Cruz Biotechnology, CA, USA) as a negative control.

Monoclonal Arc antibody (E-7 & C-7), monoclonal HSP27 antibody, monoclonal α tubulin antibody, monoclonal β-actin antibody and monoclonal GST antibody were purchased from Santa Cruz: polyclonal GAPDH antibody, monoclonal HA antibody and polyclonal Prx6 antibody from Ab Frontier, Inc.: monoclonal Hsc/Hsp70 antibody, monoclonal HSF1 antibody and monoclonal PARP-1 antibody from Enzo: monoclonal FLAG antibody from Sigma Aldrich: monoclonal Lamin B antibody from Calbiochem. Hydrogen peroxide was purchased from Samchun Pure Chemical Co. and diamide, sodium arsenite and cycloheximide from Sigma Aldrich.