Mf chemibis 3

Cells were collected and lysed in radioimmunoprecipitation assay buffer (Solarbio, Beijing, China) supplemented with phenylmethanesulfonyl fluoride (Pierce, Rockford, IL) after treatment for 48 h. Total protein concentrations were determined by bicinchoninic acid assays (Sangon Biotech). Proteins were separated on 10% polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA). After overnight blocking with 5% dried nonfat milk in PBS containing 0.1% Tween 20, membranes were incubated with mouse anti-CASR polyclonal antibody (1:500; Abcam, Cambridge, MA, US) and mouse anti-GAPDH monoclonal antibody (1:1000; Santa Cruz Biotechnology). Membranes were then washed 3 times with PBS-Tween and incubated for 1 h at room temperature with 3,500-fold diluted horseradish peroxide-labeled mouse anti-rabbit IgG (Santa Cruz Biotechnology). Immunoreactive bands were detected using the enhanced chemiluminescence detection kit (Amersham Biosciences, Little Chalfont, UK) and scanned on a chemiluminescent imaging system (MFChemiBIS3.2, DNR Bio-Imaging Systems Ltd., Jerusalem, Israel).

After the treatments, cells were directly lysed in SDS-buffer (62.5 mM Tris–HCl pH 6.8, 2% SDS, 20% glycerol, 10 µM leupeptin, 1 µg/mL pepstatin and 1 mM PMSF). Cell extracts were sonicated for 10 min on ice, centrifuged at 15,000 × g for 10 min and supernatants used to measure total protein concentration with BCA protein assay kit (Thermo Scientific-Pierce, Waltham, Massachusetts, USA). Equal amounts of total protein were loaded onto 10% or 14% SDS-PAGE for Western transfer to PVDF membranes and immunoblotting.
Immunoblots were probed with mouse anti-DJ-1 monoclonal antibody (1:1000, MBL, Woburn, Massachusetts, USA, Clone 3E8); rabbit anti-DJ-1 polyclonal antibody (1:1000, Abcam, Cambridge, UK, ab18257); rabbit anti-LonP1 polyclonal antibody (1:1000, Abcam ab103809) and rabbit anti-Tim23 polyclonal antibody (1:1000, Abcam ab230253). Mouse α-Tubulin (1:10,000, DM1A, Sigma, Darmstadt, Germany) monoclonal antibody was used as loading control. The blots were developed with a peroxidase-labeled goat anti-mouse or anti-rabbit secondary antibody (1:5000, Biorad, Hercules, California, USA) and chemiluminiscence detection MF-ChemiBIS 3.2 (DNR Bio-Imaging Systems, Neve Yamin, Israel). Blots were analyzed by quantitative densitometry using Totallab TL100 software (version 1.0, TotalLab Ltd., Newcastle upon Tyne, UK) and protein levels were normalized respect to tubulin.

For quantifying expressions of h-ApoE4, ATP6AP1, NLRP3, pro-caspase-1, caspase-1, GSDMD, ASC, UCP4, β-actin and GAPDH, the protein content was determined by the Bradford method [53 (link)], then the samples containing 100 μg of protein were added to 10% SDS-polyacrylamide gel electrophoresis. After electrophoretic separation and the gels were transferred to PVDF membranes, the samples were blocked by 5% skimmed milk powder for 1 h, and membranes were incubated overnight with the primary antibodies, specific to either human-specific ApoE4 at 1:1000 dilution, ATP6AP1 at 1:500 dilution, NLRP3 at 1:1000 dilution, pro-caspase-1 at 1:1000 dilution, caspase-1 at 1:1000 dilution, GSDMD at 1:1000 dilution, ASC at 1:1000 dilution, UCP4 at 1:1000 dilution, β-actin at 1:5000 dilution and GAPDH at 1:3000 dilution. After washing, specific binding was detected by horseradish peroxidase-conjugated secondary antibodies. Staining was visualised by ECL detection reagents and was analysed with an Electrophoresis Gel Imaging Analysis System (MF-ChemiBIS 3.2, DNR Bio-Imaging Systems, Israel). Band density was measured with Window AlphaEaseTM FC 32-bit software [54 (link)].