Clarity max western ecl substrate

NEM-alkylated redox Western-blotting was performed to detect the redox status of HSPB1 and KEAP1 as we previously reported.⁵⁹ (link) After treatment, rat H9c2 cardiomyocytes were washed twice with ice-cold phosphate buffer saline (PBS) and then precipitated with chilled 10% trichloroacetic acid for 30 min at 4°C. The homogenate was then centrifuged at 12,000×g for 10 min (4°C), the harvested protein pellets were washed twice with 100% acetone and dissolved in nonreducing buffer (100mM Tris-HCl, pH6.8; 2% SDS; and 40 mM NEM), and 5% dithiothreitol was added to the samples. 30 ∼ 50 μg protein was loaded onto a non-reducing SDS-PAGE gels and the proteins were transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk in Tris-buffered saline with Tween 20, membranes were incubated with different primary antibodies (dilution: 1:1000) at 4°C overnight, followed by HRP-conjugated secondary antibody (dilution: 1:5000) at room temperature for 1 h. Enhanced chemiluminescence (ECL) was performed using Clarity Max™ Western ECL Substrates (Bio-Rad, Inc., #1705062, Hercules, CA, USA). The relative band intensity was analyzed by Quantity One software (Bio-Rad, Inc., Hercules, CA, United States).

The whole-cell lysates prepared with Laemmli cell lysis buffer were separated on TGX FastCast acrylamide gels (BioRad, Mississauga, ON). Nitrocellulose membranes and the Trans-Blot Turbo system (Bio-Rad) were used for protein transfer. The non-specific antibody binding sites were blocked with 5% non-fat milk in TBST, pH 7.6 for 1h at RT. Antibodies and incubation conditions are listed in Table 1. Proteins were visualized using Clarity (Max) Western ECL Substrates (Bio-Rad, Cat. 1705060, Cat. 1705062) and ChemiDoc MP Imaging System (Bio-Rad). Band volume quantification (densitometry) was done in the Image Lab (Bio-Rad).

Isolated APP^NL-F and WT exosome samples were lysed with 5× RIPA buffer with Triton X-100 (AAJ62885AD, Fisher) and 25× P8340 (protease inhibitor cocktail) to a final concentration of 1× RIPA +1% P8340. Total protein amount for exosome and cortical homogenate samples was determined using a DC^™ Protein Assay Kit II (Bio-Rad) per manufacturer’s instructions. For exosome samples, 30 μL of the final lysate was loaded on 4–15% Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad). Tissue homogenates were diluted for a final loading protein content of 30 μg (20 μL loaded per lane). Separated proteins were then transferred onto a PVDF membrane (Bio-Rad) with the Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was blocked with 5% fat-free skim milk in TBST (Tris-buffered saline +0.05% Tween-20) or Super-Block^™ T20 (TBS) Blocking Buffer (Thermo Scientific) then incubated with primary antibody overnight at 4 °C (Alix [clone 1A12], 1:500, Santa Cruz; human APP [clone 6E10], 1:1000, BioLegend). Secondary antibodies, including ECL anti-mouse IgG (1:10000, GE Healthcare NA931V) are diluted with Super Blocking Buffer. Bands were visualized on Chemidoc MP imaging system (Bio-Rad) with ECL Plus chemiluminescent substrate (Thermo Fisher Scientific) or Clarity Max Western ECL Substrate (Bio-Rad).

Cell samples were lysed with RIPA lysis buffer, consisting of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40 (volume-to-volume), 0.1% sodium dodecyl sulfate (SDS), 0.25% sodium deoxycholate, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride, in the presence of protease inhibitor cocktail (catalog no. P8340, Sigma-Aldrich, St. Louis, MO) at a 1:100 dilution. The lysates were mixed with loading buffer, heated at 100°C for 10 minutes and then loaded on an SDS polyacrylamide gel. After electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes. The membranes were incubated overnight with primary antibodies ELL2 (A302-505A, Bethyl Laboratories, Montgomery, TX), STAT1 (9172S, Cell Signaling Technology, Danvers, MA), p-STAT1 (9167S, Cell Signaling Technology), p-CDK2 (2561, Cell Signaling Technology), cyclin D1 (sc-8396, Santa Cruz Biotechnology, Dallas, Texas), cyclin E (sc-247, Santa Cruz Biotechnology), or GAPDH (sc-47724, Santa Cruz Biotechnology) overnight. Following incubation with an appropriate secondary antibody, the membranes were incubated in Clarity Max Western ECL Substrate (1,705,062, Bio-Rad Laboratories, Hercules, CA) for 2 minutes, and visualized using ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories, Hercules, CA).