Immobilon western chemiluminescent hrp substrate ecl

Proteins were extracted from cells with RIPA lysis buffer (Beyotime, China) and were quantified using a BCA Protein Assay Kit (Beyotime, China). Whole cell lysates (40 μg of protein lysates for detecting p53, p21, cyclinD1 and β-actin, and 80 μg for detecting Bcl2, Bax) were run on 8 or 12 % SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). The membrane was blocked in 5 % nonfat milk and incubated overnight with the specific primary antibody at 4 °C, and further incubated for 1 h with the HRP-conjugated secondary antibody. The immunoreactive bands were visualized by Immobilon™ Western Chemiluminescent HRP Substrate (ECL) (Millipore, USA), using UVP Bioimaging system (UVP, Upland, CA).

Western blot analysis of nuclear and total protein of Nrf2 was performed as previously described with slight modifications [64 (link)]. Total cellular protein was prepared using the Cell Lysis Buffer (10×) (CST, USA), while nuclear protein was extracted using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo, USA). Samples were boiled for 10 min with SDS-PAGE (5×) loading buffer. After being loaded onto a 10% SDS-PAGE gel for electrophoresis, resultant samples were transferred onto PVDF membranes (Millipore, USA). The membranes were blocked with TBST containing 5% skim milk for 1 h at room temperature and then incubated with diluted primary antibodies overnight at 4 °C. After three additional washes (10 min each) with TBST, the blots were incubated with diluted HRP-conjugated secondary antibody (1:2500, Promega, Madison, USA) for 1 h at room temperature. β-actin and Lamin B1 were used as the internal control for total and nuclear protein expression, respectively. Signals were detected using Immobilon Western Chemiluminescent HRP Substrate (ECL) (Millipore, USA) and analyzed using Image J 1.42q software (National Institute of Health, Bethesda, MD, USA).

Protein samples were made 24 h after transfection. Cells were washed with ice-cold PBS, scraped on ice in lysis buffer (50 mM Tris–HCl pH 8.0, 50 mM KCl, 10 mM EDTA, 1 mM PMSF, 1% Triton X-100) and incubated for 16 min on ice. After centrifugation, the protein amounts of the supernatants were measured by Bradford reagent (Thermo Scientific). Samples were boiled with Laemmli buffer at 100 °C for 5 min. 20 μg protein per sample were applied on the SDS polyacrylamide gel and after electrophoresis, proteins were transferred to PVDF membranes. The following primary antibodies were used: anti-FLAG (Sigma, M2, mouse, 1:2000), anti-HA (Roche, rat, 1:1000), anti-VPS18 (Abcam, rabbit, 1:3000), anti-VPS11 (Sigma, rabbit, 1:500), anti-VPS8 (Atlas, rabbit, 1:500), anti-VPS41 (Abcam, rabbit, 1:1000), anti-Tgfbrap1 (Abcam, rabbit, 1:200), anti-LC3 (Nanotools, mouse, 1:200), anti-p62 (Medical & Biological Laboratories, rabbit, 1:1000), anti-tubulin (DSHB, mouse, 1:800). For development of specific protein bands, horseradish peroxidase (HRP) conjugated secondary antibodies were used in 1:2500 (anti-rabbit, DAKO) or in 1:10.000 (anti-mouse, Sigma) dilution. Protein detections were performed with Immobilon Western Chemiluminescent HRP Substrate (ECL, Millipore). Band intensities were analysed by ImageJ software. Supplementary Figures include unprocessed original version of western blot images.