Blot fresh western blot stripping reagent

HuCCT1 or H69 cells treated with 800 ng/mL ESPs, for the indicated times, were washed with ice-cold PBS and then lysed with a RIPA buffer containing a complete protease inhibitor cocktail (Sigma-Aldrich). Thirty μg of total soluble protein was separated by SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane (Millipore, Bedford, MA). Membranes were probed with primary antibodies against E-cadherin (1:3000 dilution) or N-cadherin (1:1000). After incubation with host-specific secondary antibodies, the immunoreaction was detected with a West-Q-chemiluminescent substrate kit (GenDEPOT, Barker, TX) and quantitated by densitometric scanning of the X-ray film with a Fluor-S Multimager (Bio-rad, Hercules, CA). The blots were normalized for protein loading by washing in Blot-Fresh Western Blot Stripping Reagent (SignaGen Laboratories, Gaithersburg, MD) and re-probing with a polyclonal antibody against GAPDH (1:5000 dilution).

Cells were washed with ice-cold PBS and then lysed with RIPA buffer (Sigma-Aldrich), followed by homogenization with a sonicator. After clearing lysates by centrifugation, total soluble protein (50 µg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels. The proteins were transferred to a nitrocellulose membrane (GE Healthcare Biosciences, Uppsala, Sweden) and probed with primary antibodies against MMP1, −2, −9 or −13 (Abcam, Cambridge, MA, USA), followed by incubation with host-specific peroxidase-conjugated secondary antibodies. Immunoreactive proteins were detected with a West-Q chemiluminescent substrate kit (GenDEPOT) and quantified by densitometric scanning of the X-ray film with a Fluor-S Multi-imager (Bio-Rad). Blots were normalized with respect to protein loading by washing in BlotFresh Western Blot Stripping Reagent (SignaGen Laboratories, Gaithersburg, MD, USA) and reprobingwith a polyclonal antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; AbFrontier Co., Seoul, Korea).