A5316

Whole-cell extracts were prepared by lysing cells in Laemmli sample buffer. Equal amounts of total protein were fractionated on SDS-PAGE, and standard protocols were applied for western blotting. Proteins were revealed with primary antibodies against HA-tag (Roche; 12CA5), FLAG (Sigma; F3165), SRSF10 (Abcam; ab77209), hnRNP F or hnRNP H (kindly provided by Doug Black), hnRNP K (kindly provided by G. Dreyfuss), actin (Sigma; A5316), tubulin (ab4074; Abcam), using peroxidase-conjugated secondary antibodies and ECL detection reagent (Amersham). Secondary antibodies were either polyclonal anti-rabbit (Cell Signaling; 7074) or anti-mouse (Bio-Can; 115-035-003).

Cell lines were grown to 70 to 80% confluence and lysed in RIPA (10% glycerol, 50 mM Tris–HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% NP40, 0.2% sodium deoxycholate, aqueous) containing protease and phosphatase inhibitors (Thermo Fisher Scientific) and benzonase (Santa Cruz). Protein concentrations were determined using the bicinchoninic acid (BCA) Protein Assay Kit, equally loaded and separated by SDS-PAGE, transferred to a nitrocellulose membrane, blocked in 5% milk, and incubated with primary antibodies overnight at 4 °C. Washed membranes were incubated for 45 min at room temperature in secondary antibody solution (LI-COR IRDye 680, 800; 1:10,000 in 5% milk), imaged on an Odyssey CLx, and analyzed using Image Studio Software (https://www.licor.com/bio/image-studio/). Antibodies were used at the following dilutions: β-actin (MilliporeSigma #A5316, 1:5000), HMOX1 (Abcam #ab13243, 1:1000), NRF2 (Cell Signaling Technology #20733, 1:1000), and NQO1 (Novus #NB200-209, 1:1000).

The following antibodies were used: β-ACTIN mouse monoclonal (Sigma-Aldrich, A5316; 1:8000 WB), LAMP-1 rabbit polyclonal (Abcam, ab24170, 1:500 IF), LAMP-1 mouse monoclonal (DSHB, H4A3; 1:500 WB), and MYC mouse monoclonal (Sigma-Aldrich, SAB2702192; 1:2000 WB; 1:200 IF).

SDS-PAGE was performed using NuPAGE 4–12% Bis–Tris gels (Thermo Fisher Scientific). After electrophoresis, proteins were transferred onto a nitrocellulose membrane (Biorad) and probed with antibodies raised against mouse anti-Parkin (Cell Signaling, mAb #4211, 1:1000), mouse anti-SLP-2 (Abcam, ab89025, 1:1000), rabbit anti-HA (Santa Cruz, sc-805, 1:500), mouse anti-β-actin (Sigma Aldrich, A5316, 1:5000), mouse anti-tubulin (Abcam, ab44928, 1:5000), mouse anti-GAPDH (Millipore, MAB374, 1:500). Immunoreaction was visualized using the SuperSignal West Dura Chemiluminescence Westernblot Substrate (Thermo Fisher Scientific).

Western blotting and immunoprecipitation were conducted as previously described [20 (link)]. The cell and tissue proteins were lysed and isolated in the RIPA lysis buffer including protease inhibitor and phosphatase inhibitors for 30 min at 4 °C. The protein was obtained after centrifugation at 12000xg for 10 min, and the protein concentration was detected and quantified by the Pierce BCA Protein Assay Kit (Thermo Scientific, #23,225). An equal amount of protein was added to the 10% SDS-PAGE gel and transferred onto PVDF membranes. The membrane was incubated with the primary antibodies overnight at 4 °C. Then the membrane was washed three times with TBST for 5 min and incubated with the secondary antibodies at room temperature for 1 h. The primary antibodies are detailed in the Additional File 1: Table S1. The secondary antibodies included the HRP-labelled goat anti-rabbit IgG (ZSGB-BIO #ZB-2301, Beijing, China) and HRP-labelled goat anti-mouse IgG (ZSGB-BIO #ZB-2305, Beijing, China). The β-actin antibody (Sigma-Aldrich #A5316) and the Lamin B antibody (Abcam #ab16048) were used to confirm equal protein loading among the samples. The signals were detected with an enhanced chemiluminescence system (West Pico kit, Pierce, Loughborough, UK). The band density was analysed using the Image J software (National Institutes of Health imaging software).