Ab270604

Proteins were obtained by RIPA lysis buffer containing fresh protease and phosphate inhibitor mixture and boiled for denaturation. Protein concentration was determined by BCA protein assay. Cell lysate was then prepared for western blotting using a 10% polyacrylamide gel and 30 μg of protein. After electrical transfer and blocking with 5% BSA, the membrane was incubated with primary antibodies at 4°C overnight. Antibodies against β-actin (A5441) were obtained from Sigma–Aldrich; antibodies against TFEB (ab270604), NLRP3 (ab263899), PINK1 (ab23707), and Parkin (ab77924) were purchased from Abcam; and antibodies against GAPDH (SC-365062), caspase-1 (SC-56036), and P62 (SC-48402) were obtained from Santa Cruz Biotechnology.

Total protein was collected from cells. Protein concentration was quantified using a BCA kit (Abcam, Shanghai). Afterwards, 30 µg of protein was separated by 12% SDS-PAGE at 120 V for 1 h. The separated protein was moved onto PVDF membranes (Millipore, Beijing), which was then sealed with nonfat milk. The membranes were incubated with primary antibodies including anti-TLR4 (ab13556, 1:500, Abcam, Shanghai), anti-TFEB (ab270604, 1:1000, Abcam, Shanghai), anti-LC3 I/II (ab128025, 1:1000, Abcam, Shanghai), anti-p62 (ab109012, 1:10000, Abcam, Shanghai), and anti-GAPDH (ab9485, 1:2500, Abcam, USA) at 4°C overnight and with secondary antibodies (ab205718, 1:10000, Abcam, Shanghai) at room temperature for 2 h. The bands were captured with an ECL kit (Abcam, Shanghai) and quantified using Scion Image v. 4.0.2 software (Scion Corporation).

The cells were washed twice with cold PBS buffer and lysed on ice with RIPA (P0013B, Beyotime, China) lysis buffer for 30 min. The lysate was centrifuged at 4°C for 15 min at 14,000 g. The supernatant was collected, and protein was extracted by the Beyotime BCA kit (P0010, Beyotime, China) to determine the protein concentration. The protein was then denatured in loading buffer and denatured by boiling in a 95°C water bath for 5 min. The protein samples (20 μg of protein per gel lane) were loaded onto SDS–PAGE gels and then transferred to PVDF membranes. The membranes were blocked in 5% bovine serum albumin (BSA) for 2 h at room temperature and incubated overnight in the primary antibody at 4°C. The antibody information is as follows: LC3 (#3868 s, Cell Signaling Technology, USA), Lamp1 (ab24170, Abcam, UK), NLRP3 (bs-10021R, Bioss, China), Caspase-1 (#3866, Cell Signaling Technology, USA), Caspase-1 (bs-0169R, Bioss, China), IL-1β (bs-0812R, Bioss, China), TFEB (ab270604, Abcam, UK), and β-actin (AA128-1, Beyotime, China). The PVDF membrane was washed and incubated with horseradish peroxidase and secondary antibody for 1 h. An enhanced chemiluminescence kit (BL520B, Biosharp, China) and gel imaging system (Bio-Rad, USA) were utilized to collect protein development images. ImageJ software was used for the quantitative analysis of signals.