Ab183896

After electrophoresis, the proteins were blotted into polyvinylidene difluoride membranes under semi-dry transfer conditions (Thermo Scientific, Rockford, IL). After blocking with 5% milk, the following antibodies were used: anti-VDR (1:200, sc-13133; Santa Cruz Biotechnology, Dallas, TX), anti-TNF-α (1:500, ab183896; Abcam, Cambridge, United Kingdom), and secondary antibodies (Code: 115-035-146; Code: 111-035-003, Jackson ImmunoResearch, West Grove, PA). Protein loading was normalized to anti-GAPDH (1:5,000, sc-365062; Santa Cruz). Bands were visualized by chemiluminescence detection (Chemiluminescent HRP Substrate, Millipore, MA) and quantified using the MicroChemi 2.0 System. Specific bands were quantified by densitometric analyses with GelQuant software (Jerusalem, Israel).

Tissues were homogenized and lysed (30 min on ice) in RIPA buffer supplemented with protease inhibitor cocktail (Sigma, USA) to harvest total protein. The homogenate was centrifuged at 14,000 × g for 15 min. Equal amounts of protein (20 μg) were loaded on 4%–20% Ready Gels (Bio-Rad Laboratories) and then transferred to a polyvinylidene difluoride membrane. After blocking with QuickBlock Western blocking buffer (Beyotime Biotechnology), the membrane was incubated with primary antibodies against MyD88 (Abcam, ab219413), NF-κB (Abcam, ab288751), TNF-α (Abcam, ab183896), IL-1β (Abcam, ab234437), and IL-6 (Abcam, ab259341) at 4°C overnight and probed with secondary antibodies conjugated with horseradish peroxidase for 1-2 h at room temperature. The bands were visualized by enhanced ECL chemiluminescence reagent (Invitrogen, USA). Signals were analyzed with Image Lab and normalized to controls.