Iblot2 transfer stacks pvdf membrane

Cells were rinsed twice, with cold PBS, and lysed in ice-cold RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0; Thermo Fisher Scientific), supplemented with complete Protease Inhibitor Cocktail (Sigma Aldrich) and PhosStop (Thermo Fisher Scientific). Protein concentrations were determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) as per the manufacturer’s protocol. Up to 30 µg of protein per sample was loaded on NuPAGE 8–12% Bis-Tris protein gels (Thermo Fisher Scientific) and transferred onto iBlot2 Transfer Stacks PVDF membrane (Thermo Fisher Scientific). Membranes were blocked with Odyssey Blocking buffer (Milennium Science, Victoria, Australia) for 1 h, probed with rabbit anti-STAT3p antibody (1:200; 9145, Cell Signaling Technology) and mouse anti-β-actin (1:5000; NB600-501, Novus Biologicals) overnight, followed by detection with goat anti-rabbit IgG DyLight 800 (1:20,000; Thermo Fisher Scientific) and goat anti-mouse IgG DyLight 600 (1:20,000; Thermo Fisher Scientific). Bands were visualized using the Odyssey CLx system (LI-COR Biosciences, Nebraska, USA) and analyzed with Image Studio Lite V5.2 (LI-COR Biosciences).

THP-1 cells were infected with BCG with/without 100 nM 7α,25-OHC and with/without 10 µM GSK682753 and lysed at 6 or 24 h post infection (p.i.) in ice-cold RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0; Thermo Fisher Scientific), supplemented with complete Protease Inhibitor Cocktail (Sigma Aldrich) (120 µl RIPA/1 × 10⁶ Cells). Protein concentrations were determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) as per manufacturer’s protocol. 10 µg of protein per sample was loaded on NovexTM 10–20% Tris-Glycine protein gels (Thermo Fisher Scientific) and transferred onto iBlot2 Transfer Stacks PVDF membrane (Thermo Fisher Scientific). Membranes were blocked with Odyssey Blocking buffer (Milennium Science, Victoria, Australia) for 2 h, probed with rabbit anti-human LC3B (1:1,000, Sigma L7543) and rabbit anti-human GAPDH (1:2,500, Abcam 9485) overnight, followed by detection with goat anti-rabbit IgG DyLight 800 (1:20,000; Thermo Fisher Scientific). Bands were visualized using the Odyssey CLx system (LI-COR Biosciences, Nebraska, USA) and analyzed with Image Studio Lite V5.2 (LI-COR Biosciences).