Mouse anti gapdh 6c5

Mouse anti-HA (F-7), mouse anti-GAPDH (6C5) and mouse anti-β-actin (C4) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The mouse anti-flag antibody and protease inhibitor cocktail were obtained from Sigma (Saint Louis, MO, USA). The MCL1 and ubiquitin antibodies were obtained from Proteintech Company. The CellTiter-Glo Luminescent Cell Viability Assay Kit and Caspase-Glo^® 3/7 Assay Kit were obtained from Promega Company. MG-132 was obtained from Merck Millipore Company. ABT-263 was obtained from Selleck Company. Sorafenib was obtained from MCE Company. The TRIZOL reagent was obtained from Invitrogen. The Top Green qPCR SuperMix was obtained from Transgen Biotech.

Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Twenty μg of podocyte cell lysate were resolved in 10% SDS-PAGE and transferred to a nitrocellulose membrane by wet blot transfer. The detection of protein bands was performed using horseradish-peroxidase-labelled secondary antibodies and visualized using enhanced chemiluminescence reagents. The primary antibodies used: mouse-anti POEM (G-1, Santa Cruz, Dallas, TX, USA), mouse-anti GAPDH (6C5, Santa Cruz), rabbit-anti bTubulin (9F3, Cell Signaling), rabbit-anti Giα3 (06-270, Sigma Aldrich, St. Loius, MO, USA), rabbit-anti Lamin A/C (#2032, Cell Signaling, Danvers, MA, USA), and mouse-anti β-Actin (sc-47778, Santa Cruz). The secondary antibodies used: anti-mouse HRP and anti-rabbit HRP (DAKO, Agilent, Santa Clara, CA, USA).
A semi-quantitative analysis of protein expression was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) by measuring band intensities of target proteins and a reference protein, as indicated. Fold expression was calculated.