Anti calmodulin

Homogenized liver tissues and samples were lysed in RIPA buffer containing a protease inhibitor cocktail (Roche) and phosphatase inhibitor (Sigma-Aldrich). Quantified protein extracts (40 μg) were loaded in 6 ~ 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The following primary antibodies were used: anti-SERCA2b (1:500; Invitrogen), anti-mitochondrial calcium uniporter (MCU) (1:1000; Invitrogen), anti-IP3R (1:800; Cell Signaling Technology), anti-voltage-dependent anion-sensitive channel 1 (VDAC1), anti-total-eukaryotic initiation factor 2α (eIF2α), anti-phospho-eIF2α, anti-activating transcription factor 4 (ATF4), anti-CHOP, anti-PI3K-p110α (1:1000; all from Cell Signaling Technology), anti-PI3K-p85 (1:3000; BD Biosciences, San Jose, CA, USA), anti-PERK (1:200; Santa Cruz), anti-GRP75 (1:500; Abcam), and anti-calmodulin (1:500; Novus Biologicals). The loading control was anti-GAPDH (1:2000; AbFrontier, Seoul, Republic of Korea). After the membranes were washed, the following secondary antibodies were used: anti-mouse IgG and anti-rabbit IgG (1:8,000; all from Bio-Rad). The protein bands were detected using a Clarity Western ECL kit (Bio-Rad) and a ChemiDoc imaging system (Bio-Rad).

To determine calcium channel expression in the ER, liver tissues from each group were sectioned at a thickness of 5 μm and fixed with 10% NBF. The fixed tissues were reacted in 3% hydrogen peroxide (H₂O₂) in 100% methanol to block endogenous peroxidase activity. The following antibodies were used: anti-CHOP (1:50; Santa Cruz, Dallas, TX, USA), anti-calmodulin (1:100; Novus Biologicals, Littleton, CO, USA) and anti-PCNA (1:500; company). CHOP was performed using Proteinase K (20 μg/mL) (Dako). However, anti-calmodulin and anti-PCNA were used for antigen retrieval before incubation at 4 ℃ overnight. Incubation with horseradish peroxidase-conjugated streptavidin–biotin complex (Dako) and 3,3-diaminobenzidine (EnVision™ Systems, Santa Clara, CA, USA) was performed to generate a chromatic signal. For counterstaining, Mayer’s hematoxylin (Dako) was used. Representative images were captured and quantified using a digital slide scanner (3DHISTECH, Ltd.).