Af0246

Cells were harvested and lysed with RIPA buffer (Beyotime, Jiangsu, China) followed by BCA analysis to determine the protein concentration. The samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes. After incubation with the primary antibodies anti-p65 (1:1000; AF0246, Beyotime, Jiangsu, China), anti-phosphor p65 (1:1000; AN371, Beyotime, Jiangsu, China), anti-GAPDH (1:10000; 10494–1-AP, Proteintech Group, Illinois, USA), anti-LAMB1 (1:5000; 23498–1-AP, Proteintech Group, Illinois, USA), the membranes were then incubated with a secondary antibody (1:5000; #7074, Cell Signaling Technology, Massachusetts, USA). After washing, signals were detected using a chemiluminescence system (Bio-Rad, California, USA) and analyzed using Image Lab Software (Bio-Rad, California, USA).

For these assays, 14 mm round cover slides were placed in each well in a 24-well plate for hepatocytes to adhere to the wall. The cells were fixed with 4% paraformaldehyde (300 μL/well, P6148, Sigma-Aldrich) for 15 min, washed with PBS 3 times (5 min each), and perforated with 0.3% Triton X-100 (500 μL/well, T9284, Sigma-Aldrich) for 15 min to improve cell permeability. The cells were washed again with PBS three times, and then the sealant (3% BSA) was added to seal the samples in the incubator at 37 °C for 1 h. Then, primary antibodies p65 (1:100, AF0246, Beyotime), STIM1 (1:200, AF2614, Beyotime), Orai1 (1:200, 66223-1-Ig, ProteinTech), and Calnexin (1:100; AC019, Beyotime) were added, and the mixture was incubated overnight at 4 °C. After the cells were washed with PBS three times, the fluorescent secondary antibodies (1:500, A0562, and A0568, Beyotime) were added and incubated at 37 °C for 1 h without light. After three washes with PBS, the nuclei were stained with a DAPI solution (D8417; Sigma-Aldrich), and a round glass cover was fixed on the glass slide after washing for three times in the dark. The cells were observed by an LSM 710 confocal laser microscope system (Zeiss, Oberkochen, Germany).

Cells were lysed in RIPA lysis buffer (Beyotime), separated by SDS‐PAGE, and transferred onto PVDF membranes (Millipore). Membranes were blocked with 5% defatted milk for 1 h and incubated with anti‐ICAT (A7122, 1:2000, ABclonal), anti‐ICAT (ab129011, 1:2000, Abcam), anti‐JUP (381,616, 1:2000, ZEN‐Bioscience), anti‐GAPDH (10494‐AP, 1:5000, Proteintech Group), anti‐phospho‐P65 (p‐P65) (AF5881, 1:1000, Beyotime), or anti‐P65 (AF0246, 1:1000, Beyotime) primary antibodies at 4°C for 10 h. The membranes were washed thrice with TBST for 5 min each and incubated with horseradish peroxidase‐conjugated secondary antibodies to rabbit (#820112, 1:2000, EarthOx) or mouse (#620112, 1:2000, EarthOx) for 1 h. The signals were detected using a chemiluminescence system (Bio‐Rad).