Anti grp78 antibody

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.

Co-immunoprecipitation was performed as described previously [20 (link)] with minor modifications. Briefly, cells were cultured in T175 flasks for 3 days under normal conditions until the cells reached around 80% confluence. The membrane fraction enriched protein lysate described in the earlier section was incubated with monoclonal antibody anti-GEP antibody A23 (Versitech) [17 (link)] or anti-GRP78 antibody (Cell Signaling), respectively, at a ratio of 400 μg to 2 μg. This mixture was incubated at 4 °C overnight under rotation. Antibody alone and protein lysate alone, respectively, were performed as independent control reactions and served as references for the non-specific bindings with Protein G Sepharose. For each reaction, 100 μl of equilibrated protein G-Sepharose beads (Amersham Biosciences, Piscataway, NJ) were added to the antibody-lysate mixture and incubated at 4 °C with rotation for an hour. The complexes were briefly centrifuged after incubation. The supernatant was discarded and the beads were washed with 500 μl lysis buffer for 5 times to remove any unbound proteins. The co-immunoprecipitated proteins were eluted by adding SDS sample buffer to the protein G-Sepharose beads followed by 5 min incubation at 95 °C.

Cells in culture plates were washed in ice-cold PBS twice and lysed in RIPA buffer (Cell Signaling Technology, #9806) containing complete protease inhibitor cocktail tablets (Roche). Protein lysates (20–40 μg) were resolved using SDS/PAGE and transferred to nitrocellulose membranes (Millipore). Primary antibody incubations were performed at 4 °C overnight using a 1:1000 dilution for anti-VCAM-1 antibody (ab134047, Abcam), anti-ICAM-1 antibody (ab171123, Abcam), or anti-GRP78 antibody (#3177, Cell Signaling Technology). Secondary antibody incubation was performed using a 1:5000 dilution of goat anti-rabbit HRP conjugate antibody (#7074, Cell Signaling Technology). Protein bands were visualized by LumiGLO® Reagent (#7003, Cell Signaling Technology).