Anti hsp 72

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.

Unless stated otherwise, all reagents were obtained from regular commercial sources as mentioned in the methods descriptions. The following antibodies were used: anti-BAG3 (Proteintech Group, 10599-1-AP), anti-FLAG (Sigma-Aldrich, F1804, St. Louis, MO, USA), anti-HSP72 (Enzo Life Sciences, Farmingdale, NY, USA, ADI-SPA-810), HRP anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA, 715-035-151), Cy3 anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, 715-165-151), HRP anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, 711-035-152), Alexa Fluor 647 anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, 711-605-152), anti-rabbit IgG (Sigma-Aldrich, I5006), anti-tubulin (Sigma-Aldrich, T9026), and anti-YES1 (Sigma-Aldrich, HPA026480).

Total cell extracts (50μg) from Beta-TC-6 cells were isolated according to standard protocol (Cell Signaling Technology, Danvers, MA); fractionated by electrophoresis on 12% SDS polyacrylamide gels; electroblotted to PVDF membrane (GE healthcare, Piscataway, NJ) and probed with anti-Hsp72 (Enzo Life Sciences, Farmingdale, NY), and anti-Hsp25 (Enzo Life Sciences) antibodies. To detect IAPP expression, membranes were probed with a polyclonal antibody raised against amino acids 40–89 of IAPP precursor of human origin (Santa Cruz Biotechnology, Dallas, TX) that cross reacts to a lesser extent with mouse IAPP. Protein loading control used was β-actin (Abcam, Cambridge, MA). Appropriate horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used in the study. Protein bands were visualized by chemiluminescence (Thermo Scientific, Rockford, IL), films were developed and image processor Quantity One (Version 4.6) was used to scan and to compare expression levels.