W4018

Samples were cultured in defined medium at special time. The cells extracts were subjected to SDS-PAGE and western blotting. The specific antibody, anti-GFP (MBL598), anti-Tubulin (MBL PM054), anti-Rabbit IgG (PROMEGA W4018) was used to enhance the luminescence intensity of the substrate. The protein imprint was detected by Western Blotting exposure meter (Tanon, 5200, multi).

Protein extracts were prepared by lysing cells with RIPA buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM Na₃VO₄, 1 mM NaF, 0.1 mM PMSF, 10 μM aprotinin, 5 μg/mL leupeptin, 1 μg/mL pepstatin A). Protein samples were diluted using 2x Laemmli loading buffer, mixed, and boiled for 5 minutes at 95°C. Samples were analyzed by SDS/PAGE using a 10% acrylamide gel, followed by transfer onto PVDF membranes (Millipore IPVH00010). Membranes were blocked with 5% BSA (ThermoFisher) in Tris-buffered saline (50 mM Tris-HCl pH 7.5, 150 mM NaCl) and 0.1% Tween-20 for 1 hour at room temperature. Primary antibody labeling was done with anti-IRF3 (1:1,000) (Cell Signaling 4302S), anti-P-IRF3 (1:2,000) (Cell Signaling 4947S), anti-STAT1 (1:1,000) (EMD Millipore 06–501), anti-P-STAT (1:1,000) (Cell Signaling 7649), anti-IκB (1:1,000) (Santa Cruz sc-371), anti-P-IκB (1:1,000) (Cell Signaling 2859), anti-actin (1:10,000) (Sigma A2066), or anti-human IRF3 (1:500) [48 ] overnight at 4°C. Secondary antibody labeling was done using anti-rabbit or anti-mouse IgG-HRP conjugate (1:10,000) (Promega W4018, W4021) by incubating membranes for 2 hours at room temperature. Blots were imaged onto film using luminol enhancer (ThermoFisher 1859675).

Whole cell lysates were isolated using 150 mM NaCl, 1% NP-40, 0.1% SDS, 100 mM Tris-HCl, pH 7.4, supplemented with protease inhibitors (Roche) and cleared by centrifugation at 4 °C for 10 min at 10,000 × g. Proteins lysates were separated by 4–12% SDS PAGE and then transferred to nitrocellulose membranes. The membranes were probed with monoclonal rabbit anti-SR-BI (Abcam, ab180383, 1:1,000), mouse monoclonal anti-ABCA1 (Abcam, ab18180, 1:1,000), rabbit polyclonal anti-ABCG1 (Novus Biologics, NB400-132, Lot F3, 1:1,000 dilution), or monoclonal mouse anti-GAPDH (Sigma, G8795, 1:10,000) in TBS-Tween20 containing 5% non-fat dry milk. HRP-conjugated secondary antibodies we used: anti-mouse (Promega, W4028, Lot 0000214819, 1:15,000) and anti-rabbit (Promega, W4018, Lot 0000212738, 1:20,000). Immune complexes were detected with Western Lightning Plus-ECL (Perkin Elmer) or Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) chemiluminescent substrate.