Monitoring Protein Phosphorylation and Expression in Transfected Cells

Whole protein extracts prepared from HEK-293 cells transfected with 10 µg of either pCMV-DNAJB3, pCMV or pCMV-HDAC4 vectors were used to monitor the changes in the phosphorylation levels of JNK (P-JNK) in response to 5 µM PMA stimulation by western blot essentially as we described previously^{23 (link)}. The expression of DNAJB3 and HSP-72 in whole cell extracts prepared myoblasts and myotubes was also performed by western blot using anti-DNAJB3 (Proteintech Group, Inc., Chicago, IL) and anti-HSP-72 (ENZO Life Sciences, Inc., Farmingdale, NY) antibodies. The endogenous expression of Glut4 in C2C12 overexpressing DNAJB3 (or control vector) was monitored by western bot using anti-Glut4 antibody (Abcam, Cambridge, UK). Nuclear translocation of p65 NF-κB in C2C12 transfected with DNAJB3 or pCMV after LPS/TNF-α stimulation was carried out on cytoplasmic and nuclear fractions by western blot using anti-p65 antibody (Cell Signaling Technology, Inc., Danvers, MA). Anti-GRP78 antibody (Cell Signaling Technology, Inc., Danvers, MA) was used to monitor the expression of GRP78 protein in response to Tunicamycin treatment using whole cell extracts from C2C12 transfected with DNAJB3 or pCMV. GAPDH, β-Actin (Cell Signaling Technology, Inc., Danvers, MA) and γ-Tubulin (Acam, Cambridge, UK) were used as internal controls as indicated in the figure legends.