Western Blot Analysis of Protein Expression

For Western blot analyses the cell lysates were prepared and fractionated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using the Mini-PROTEAN III Gel Electrophoresis System (BioRad, Hercules, CA, USA). A commercial mouse monoclonal anti-myc (sc-40, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as the primary antibody, diluted at 1:1000. Horseradish peroxidase-conjugated antibodies against mouse IgG (#32439, Invitrogen) were diluted to 1:1000–1:4000. Chemiluminiscence detection and the densitometry for protein band quantification was performed as previously described [36 (link)]. As an additional sample loading control, the endogenous lactate dehydrogenase (LDH) protein was detected in cell extracts using a goat anti-LDH antibody diluted to 1:5000 (AB1222, Chemicon, Temecula, CA, USA) and an anti-goat IgG horse-radish peroxidase-conjugated antibody (sc-2033, Santa Cruz Biotechnology, diluted to 1:2000). RFP (transfection control) was detected using a rabbit anti-RFP antibody (#AB233, Evrogen, Moscow, Russia), diluted to 1:5000, and an anti-rabbit IgG horse-radish peroxidase-conjugated antibody (#1858415, Pierce), diluted to 1:1000.