Immobilon western chemiluminescent hrp substrate

Proteins were separated by electrophoresis on 12% (w/v) polyacrylamide gels (SDS-PAGE), and transferred to polyvinylidene fluoride membranes (Immobilon; Millipore Corporation, Bedford, MA) in either a semi-dry or a wet blotting device (Biometra, Göttingen, Germany; Bio-Rad, Hercules, CA). After blotting, membranes were washed once in Tris-buffered saline (TBS), pH 7.4, containing 0.1% (v/v) Tween-20 (TBS-T), and incubated for blocking in TBS-T, containing 5% (w/v) dried skimmed milk for 60 min at room temperature (RT), followed by overnight incubation with a polyclonal Rab31-directed rabbit antibody (RT3-IgG; [Grismayer et al. 2012 (link)]) or with a polyclonal goat antibody directed against the so called latent-associated protein (LAP) domain of human TGF-ß1 (R&D Systems Wiesbaden, Germany), diluted in blocking buffer. After washing with TBS-T, the secondary peroxidase-conjugated goat anti-rabbit or rabbit anti-goat IgG (Jackson ImmunoResearch Lab, West Grove, PA) diluted in TBS-T, containing 1% (w/v) milk powder was applied for 60 min at RT followed by three washes with TBS-T. Proteins were visualized by a chemiluminescent reaction using ECL reagents or Immobilon Western Chemiluminescent HRP Substrate according to the manufacturers' recommendations (Thermo Scientific).

Cells were dissolved in RIPA lysis buffer supplemented with 20 mM HEPES and 0.5% Triton X-100, pH 7.6. Proteins were separated and transferred by electrophoresis to the nitrocellulose membranes (GE Healthcare life Science, Pittsburgh, PO, USA). The membranes were blocked with 5% non-fat milk containing 0.02% sodium azide, and then reacted with a primary and secondary antibody step by step. After washing with 1 X tris buffered saline with tween, images of protein band were captured by immobilon Western chemiluminescent HRP substrate and iBrightTM Imager (Thermo Fisher Scientific, Waltham, MA, USA).

The treated cells were washed with PBS and lysed in a RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with a protease inhibitor cocktail (Boster, Wuhan, China) to extract total proteins. The membrane protein was extracted from the cells using a Membrane Protein Extraction Kit (Beyotime, Shanghai, China). Immunoblotting analysis was performed as previously described (Yin et al., 2016 (link)) following standard procedures and using the primary antibodies, ABCA1, LDLR (Abcam), LXR (ABclonal), and Anti-β-actin (ProteinTech). In brief, the protein was separated on an 8–10% SDS denatured polyacrylamide gel and then transferred onto a NC membrane. The membranes were blocked with 5% skim milk and were incubated with appropriate antibodies at 4°C overnight. Then, the membranes were washed and incubated with horseradish peroxidase–conjugated secondary antibodies (1:3,000; Sanjian, Tianjin, China). The protein of interest was visualized using an Immobilon Western Chemiluminescent HRP Substrate (Thermo Fisher Scientific).