Gel pro analyzer

16HBE, HEK293T and A549 cells were preplated into six-well plates at a density of 5 × 10⁵ cells per well. The next day, the cells were inoculated with SARS-CoV-2 (MOI = 0.5), transfected with 3 μg of plasmid (using FuGENE HS transfection reagent) or treated with poly (I:C). After collection at the appropriate time points, total cellular protein was extracted with an RIPA (Thermo Scientific, Waltham, MA, USA) buffer consisting of 25 mM Tris·HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS complete EDTA-free protease inhibitors. Following SDS–PAGE, Transfer of nitrocellulose membrane by wet rotary film meter (GenScript, Nanjing, China) under the following common conditions. The membranes were blocked at room temperature for 1 h and incubated with primary antibodies diluted with 5% skim milk overnight at 4 °C, washed with 0.1% PBST, incubated with secondary antibody diluted with 5% skim milk at room temperature for 1 h, and washed with 0.1% PBST. ECL chemiluminescence was examined using a BIO-RAD (BIO-RAD, Hercules, CA, USA) imager, and the grayscale results were analyzed using a Gel-Pro analyzer.

P14 BTBR and WT mice were decapitated, and their cerebella were dissected in ice-cold PBS. The total protein was extracted immediately, and protein concentrations were measured using a Bicinchoninic Acid Kit (Beyotime, China) as previously described (Xiao et al., 2017 (link)). The total protein (20 μg) of each sample was separated by 10% SDS-polyacrylamide electrophoresis (80 V, 100 min) and then transferred to a polyvinylidene fluoride (PVDF) membrane (220 mA, 60 min). The membranes were washed in 1% Tween-20/Tris-buffered saline (TBS) (TBS-T), blocked in 5% BSA/TBS-T (RT, 2 h), and incubated with a primary antibodies (4°C, 12 h) (1) mouse anti-CB (1:2000, Swant, Switzerland) and (2) mouse anti-GAPDH (1:2000, Cell Cwbio, China), followed by peroxidase-conjugated goat anti-mouse secondary antibody IgG (1:1000, Santa Cruz Biotechnology, United States). Bands were visualized using the chemiluminescence detection kit (Pierce, United States) under a Gel-Pro analyzer (Bio-Rad Laboratories, United States). Band intensity was quantified in Image Lab (Bio-Rad Laboratories, United States), and calbindin protein was normalized to GAPDH.

Total protein was extracted using SDT lysis buffe and measured using a BCA assay kit (Pierce Biotechnology, Waltham, MA, United States). Equal amounts of proteins were separated by 6–12% polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. The membrane was cut into several strips, blocked with 5% bovine serum albumin, and probed with indicated primary antibodies at 4°C overnight (Supplementary Table S3). After incubated with horseradish peroxidase-conjugated secondary antibodies, the antigen–antibody complexes were visualized using enhanced chemiluminescence plus reagent (Millipore, Billerica, MA, United States). Experiments were carried out in triplicate. Gel-Pro Analyzer (Bio-Rad) was used for band densitometry, using β-actin as an internal reference. Relative quantification of protein levels was expressed as fold change relative to the control.