Anti mouse phospho nf κb p65

RAW264.7 cells (6 × 10⁵/well) were seeded into 24-well plates and treated with DMEM media only or 5 μg/mL of LPS or with indicated concentrations of compounds for 24 h. Cells were lysed in sodium dodecyl sulfate (SDS) sample buffer containing protease inhibitor cocktail (Roche Life Science, Mannheim, Germany). The resulted proteins were resolved by SDS-polyacrylamide gel electrophoresis and then subjected to the following antibodies: anti-mouse IKK-α, anti-mouse phospho-IKK-α, anti-mouse IκB-α, anti-mouse NF-κB p65, anti-mouse phospho-NF-κB p65 antibodies (Cell Signaling Technology, Beverly, MA), and HRP-conjugated mouse anti-GAPDH (Kangcheng, Shanghai, China). Signals were detected with an HRP-conjugated anti-rabbit IgG (Bio-Rad, Richmond, CA, USA) using an ECL system (Amersham Biosciences, Buckinghamshire, UK). The uncropped and unprocessed scans of indicated blots were supplied as Supplementary Figure 99 in the Supplementary Information.