Goat anti rabbit igg horseradish peroxidase conjugated secondary antibody

The cells were lysed with RIPA buffer (Thermo Fisher Scientific, United States). A BCA protein assay kit (Thermo Fisher Scientific, United States) was used to detect the concentration of total proteins. Equal protein (50 μg) was loaded on a 10% SDS gel and transferred to polyvinylidene fluoride membranes. Subsequently, membranes were blocked in 5% milk in TBST for 2 h. Primary antibodies against PERK (1:2,000; Genetex, United States), XBP1 (1:2,000; Sigma, CA, United States), ATF6 (1:2,000; Sigma, CA, United States), CHOP (1:2,000; Sigma, CA, United States), Caspase-12 (1:2,000; Sigma, CA, United States), JNK (1:2,000; Sigma, CA, United States), GRP78 (1:5,000; Sigma, CA, United States), p-eNOS (1:5,000; Cell Signaling, MA, United States), CaMKII (1:5,000; Genetex, United States), P-gp (1:5,000; Sigma, CA, United States), MMP9 (1:5,000; Sigma, CA, United States), and β-actin (1:5,000; Abcam, MA, United States) were utilized to incubate membranes at 4°C overnight. Membranes were washed and incubated with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:5,000; Proteintech, China) for 2 h. Western blot bands were analyzed by Bio-rad Image Lab (v5.2.1).

A498 cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer. Equal amounts of cellular protein were loaded per lane, subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently transferred to a polyvinyl difluoride (PVDF) membrane (CAT#: GVWP02500, Millipore, Billerica, MA, USA). The membrane was then blocked with 5% skimmed milk for 1 h and immunoblotted with primary antibodies specific to SLC14A1 (1: 100 dilution, CAT#: AV48116; Sigma-Aldrich, St. Louis, MO, USA) and actin (1: 5,000; CAT#: A5441; Sigma-Aldrich, St. Louis, MO, USA) at 4 °C overnight. The blot was then incubated with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1: 5,000; CAT#: SA00001-2; ProteinTech Group, Chicago, IL, USA). An enhanced chemiluminescence (ECL) western detection system (CAT#: 7003; Cell Signaling Technology, Beverly, MA, USA) was used to detect immunoreactive bands.