Grp78

Cell lysis and immunoblotting were carried out as described [31 (link)]. Specific proteins were detected using antibodies against HSP70 (GeneTex), GRP78 (610979, BD Biosciences, San Jose, CA, USA), HSC70 (sc-7298, Santa Cruz Biotechnology, CA), CEP215 (Bethyl), γ-tubulin (T6557, Sigma), EB1 (E3406, Sigma), GM130 (610822, BD Biosciences), FLAG (F3165, Sigma), respectively, and GAPDH (GTX100118, GeneTex) for use as loading controls.

Cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer containing a cocktail of protease (Roche) and phosphatase inhibitors (Roche), separated by SDS-PAGE, and transferred to Immobilon-P membranes (Millipore). The following antibodies were used: tubulin (Millipore), cleaved caspase 3 (Asp 175) (Cell Signaling), cleaved PARP (Asp 214) (Cell Signaling), total PARP (Cell Signaling), total JNK (Cell Signaling), phospho-JNK (T183/Y185) (Cell Signaling), total p38 (Cell Signaling), phospho-p38 (T180/Y182) (Cell Signaling), total MKK4 (Cell Signaling), phospho-MKK4 (Ser257/Thr261) (Cell Signaling), GRP78 (BD Biosciences), total eif2a (Cell Signaling), and phospho-eif2a (Cell Signaling), XBP-1 s (Cell Signaling), CHOP (Cell Signaling), and Kir6.2 (Santa Cruz). Quantification of western blot analysis was done using ImageJ.

Cells were treated with IT-139 alone or in combination with tunicamycin or thapsigargin for 16 hours. Cell lysates were prepared and subjected to 10% or 12% SDS-PAGE and Western blot analysis. The following primary antibodies were used: GRP78 (1:1000, BD Biosciences #610978); β-actin (1:5000, Sigma-Aldrich, #A5316); eIF2α (1:1000, Cell Signaling #2103); phospho- eIF2α (1:1000, Cell Signaling #9721). The secondary antibodies used were as follows: horseradish peroxidase conjugate goat anti-mouse (1:1000, Santa Cruz Biotechnology #sc-2005) and anti-rabbit (1:1000, Santa Cruz Biotechnology #sc-2004).

Retinas were dissected 1 day after OAB. Whole protein was extracted from mice retinas with lysis buffer (KeyGen Biotech, Nanjing, China) including protease and phosphatase inhibitors. The protein concentrations were quantified with a Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein from each sample were loaded into lanes on a 10% sodium dodecyl gel for polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 5% skim milk in Tris-buffered saline containing Tween (TBST) for 2 h and then incubated with primary antibodies against IRE1 (1:1000, Abcam), GRP78 (1:1000, BD Biosciences, Franklin Lakes, NJ, USA), CHOP (1:1000, Abcam), or β-tubulin (1:1000, Cell Signaling, Danvers, MA, USA) at 4 °C overnight. Then, the membranes were washed three times with TBST and incubated with secondary antibodies (1:5000, Abcam) for 1 h at room temperature. The protein bands were visualized on a ChemiDoc Touch Imaging System (Bio-Rad), and band intensity was analyzed using ImageJ software.

Western blot analysis was performed as described previously [37 (link)]. Briefly, 30 μg of protein were run on 10% SDS-PAGE, transferred to nitrocellulose membrane (BioRad) and immunoblotted with the following antibodies: GRP78 (1:1000 BD Bioscience, San Jose, CA, USA), IGFBP-3 (1:1000 Santa Cruz, Dallas, TX, USA) and α Tubulin (1:5000 Merck Millipore, Burlington, MA, USA). After incubation with specific secondary antibodies conjugated to peroxidase (Sigma, St. Louis, MO, USA), proteins were visualised by Clarity ECL substrate (BioRad) using BioRad Chemidoc XRS + system and analysed using Image lab software (BioRad).