Ab0035

Equal amounts of total protein were separated by SDS-PAGE and transferred to PVDF membranes (Merck Millipore, Billerica, MA, USA). The membranes were incubated with 5% skim milk for 1 h at 37 °C, washed with TBST, and then probed for 2 h at 37 °C with antibodies targeting CD63 (1:5000 dilution, Biolegend, CY5253, USA), TSG101 (1:5000 dilution, Biolegend, CY5985, USA), S100A4 (1:1000 dilution, Abcam, ab124805, USA) and β-actin (1:3000 dilution, Abways, AB0035, China). Then, the membranes were incubated with HRP-labeled anti-rabbit IgG (1:5000 dilution, Abways, AB0101, China) for 1 h at 37 °C. After washing, the coloration was detected with 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich, St. Louis, MO, USA) or using an enhanced chemiluminescent kit (New Cell & Molecular Biotech Co., Ltd, Suzhou, China).

Total protein from tissue lysates of iPSCs or skin tissues was isolated using Radio-immunoprecipitation assay buffer (#89901; Thermo Fisher Scientific) containing 1% protease-inhibitor cocktail and 1% phenylmethylsulfonyl fluoride according to the manufacturer’s instructions. Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA, USA). The membrane was blocked for 1 h in 10% skim milk and then incubated with primary antibodies against integrin β1 (1:100; #sc-374429; Santa Cruz Biotechnology, Dallas, TX, USA) and β-actin (1:10,000; #AB0035; Abways, Shanghai, China) overnight at 4 °C. The following day, the membrane was washed three times for 10 min each with PBS containing 0.1% Tween-20 and then incubated with Peroxidase-AffiniPure donkey anti-rabbit IgG (H + L) (1:2000; #711-035-152; Jackson ImmunoResearch) at 37 °C for 1 h, followed by three washes for 10 min each with PBS. The immunoreactive bands were visualized using a chemiluminescence substrate detection system (Tanon, Beijing, China).

Aβ₄₀ and Aβ₄₂ in APP/PS1 mouse hippocampus and cortex were extracted as previously reported (Lazarov et al., 2005 (link)), for ELISA measurement of human Aβ₄₀ and Aβ₄₂, the frozen hippocampal and cortical tissue (400 mg) stored in −80°C were homogenized in 1 ml 2% SDS (dissolved in PBS), then centrifuged at 1,20,000 g for 60 min at room temperature. The supernatant was collected as the soluble fraction and quantified with human Aβ ELISA kits according to the users’ guidelines (ExCell Bio). Total Aβ levels in HEK293/APPswe cell culture medium were also quantified with ELISA.
Supernatant is also used for Western blot analyses. Proteins in supernatant were separated by SDS-PAGE, and transferred onto membrane. Proteins were labeled with β-actin (AB0035, Abways) and IL-1β rabbit polyclonal antibody (16806-1-AP, Proteintech) and the immunoreactive bands were detected by chemiluminescent detection (Bio-Rad) of peroxidase-conjugated antibody (M21002, Abmart). The intensity of each band was quantified by ImageJ and normalized to β-actin.

The Western blotting was performed following the method described previously [36 (link)]. Liver total proteins were obtained by using the ice-cold RIPA lysis buffer supplemented with protease inhibitors (Beyotime Biotechnology, China), and their concentrations were determined following the protocol of total protein quantification assay (New Cell & Molecular Biotech, China). The supernatant protein was mixed with 5× SDS loading buffer and boiled for 15 min. The obtained protein samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. Immunoblots were further incubated with 5% BSA in phosphate-buffered saline with Tween 20 (TBST) buffer. The target proteins of membrane were incubated with primary antibodies (anti-ACAT1, 1 : 800, A13273, ABclonal; anti-ACAT2, 1 : 1000, A1399, ABclonal; anti-CYP7A1, 1 : 800, A10615, ABclonal; anti-CPT1a, 1 : 800, 15184-1-AP, Proteintech; anti-ACLY, 1 : 800, ab40793, Abcam; ACOX1, 1 : 800, 10957-1-AP, Proteintech; ACOX2, 1 : 800, A12796, ABclonal; anti-α-Tubulin, 1 : 1000, M-1051-1, Huabio; and anti-β-ACTIN, AB0035, Abways) overnight at 4°C. After washing in TBST, bolts were incubated with secondary antibodies for 1 h at room temperature. The protein images were visualized by using an Odyssey CLx Imager (Licor, USA).

For western blotting, kidney tissues or podocytes were extracted and quantified. The protein samples were then boiled at 95 °C for 10 min and separated on a 6–12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, and subsequently transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was then incubated with primary antibodies overnight at 4 °C, followed by incubation with the corresponding secondary antibodies for protein expression visualization. Primary antibodies were used as follows: NPHS2 antibody (1:500, 20384-1-AP, Proteintech), phosphorylated Nephrin antibody (1:10000, ab80299, Abcam), Active Caspase-3 antibody (1:500, A11021, ABclonal), WT1 antibody (1:1000, A2446, ABclonal), β-actin antibody (1:3000, AB0035, Abways), Bax antibody (1:500, 380709, ZENBIO), RAC1 antibody (1:1000, ab155938, Abcam), β-tubulin antibody (1:3000, AB0039, Abways), OLR1 antibody (1:500, 11837-1-AP, Proteintech), SR-A1 antibody (1:500, 382017, Zenbio), CD36 antibody (1:500, 381350, Zenbio), IGF-1R antibody (1:50, sc-81464, Santa Cruz).

The cell lysates were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (ISEQ00010, Millipore). Subsequently, the membranes were blocked with 5% skimmed milk and incubated overnight at 4 °C with primary antibodies. The following antibodies were used: anti-SIRT5 (1:1000 dilution; 8782 S, Cell Signaling Technology, Danvers, MA, USA), anti-HINT1 (1:1000 dilution; 67583-1-Ig, Proteintech, Rosemont, IL, USA), anti-Succinyllysine (1:1000 dilution; PTM-401, PTM Bio, Hangzhou, China), anti-Acetyllysine (1:500 dilution; PTM-105RM, PTM Bio), anti-GFP (1:5000 dilution; 50430-2-AP, Proteintech), anti-Flag (1:5000 dilution; 80010-1-RR, Proteintech), and anti-β-Actin (1:5000 dilution; AB0035, Abways, Shanghai, China). Then PVDF membranes were incubated with HRP-conjugated goat anti-rabbit or mouse antibodies (1:5000 dilution; AB0101 and AB0102, Abways) and thoroughly washed. The protein bands were visualized using the ECL chemiluminescence detection kit (WBKLS0500, Millipore), and band intensity was quantified using ImageJ software.