Functional enzymes or structural proteins must be properly folded (i.e., have proper conformation). Most membrane and secretory proteins are properly folded in the ER, but, under stressful conditions, these proteins are often misfolded. The misfolded proteins induce the so-called ER stress response, and ER stress activates well-defined protective and death pathways. We determined the expression of ER stress-associated proteins, glucose-regulated protein 78 (GRP78)/BiP, and C/EBP-homologous protein (CHOP) by immunoblotting. RGC-5 cells, cultured under different conditions with or without the addition of SNAP (100 μM), were lysed using a cell lysis buffer (R0278) with protease (P8340), phosphatase inhibitor cocktails (P2850 and P5726; Sigma-Aldrich), and 1 mM EDTA. Cell lysates were solubilized in SDS sample buffer, separated on 10% SDS-polyacrylamide gels, and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA). The transfers were blocked for 1 hour at room temperature (5% Blocking One-P; Nakarai Tesque, Kyoto, Japan) in 10 mM Tris-buffered saline with 0.05% Tween 20 (TBS-T) and incubated overnight at 4°C with the primary antibody. The transfers were then rinsed with TBS-T and incubated for 1 hour at room temperature in horseradish peroxidase-conjugated goat anti–rabbit or goat anti–mouse antibody (Pierce, Rockford, IL) diluted 1:2000. The immunoblots were developed using chemiluminescence (Super Signal West Femto Maximum Sensitivity Substrate; Pierce) and were examined with a digital imaging system (FAS-1000; Toyobo, Osaka, Japan). The primary antibodies used were mouse anti-BiP (BD Bioscience, San Jose, CA), mouse anti-CHOP (Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit anti-actin (Santa Cruz Biotechnology).