Anti bid antibody

Differentiated U937 and THP-1 cells were challenged with FA1090 at an MOI of ~100 and plates were centrifuged at 1500 rpm for 4 min. After 3–4h of infection, the cells were treated with DMSO or TNF-α 10 ng/ml and cycloheximide 200ng/ml for an additional 3h. Cells were washed with PBS and lysed with RIPA buffer for 30 min at 4°C under rotation. Lysates were centrifuged at 12,000 rpm for 10 min at 4°C. The supernatants were transferred to fresh tubes and stored at −80°C. 7 to 23 μg of cellular extracts in SDS sample buffer containing β – mercaptoethanol were separated on 15% SDS-polyacrylmide gel and proteins were transferred to a polyvinylidene fluoride membrane in 10 mM CAPS (N-cyclohexo-3-amino-propanesulfonic acid) buffer with 10% methanol at pH 11 for 1h. After saturation in Tris buffered saline containing 0.1% Tween 20 and 5% dry milk, membranes were incubated overnight with anti-Bid antibody (1:1000, #2002, Cell Signaling) or anti-α-β-tubulin antibody (1:1000, 9F3, Cell Signaling). The membranes were then washed and incubated with secondary goat anti-rabbit antibody conjugated to horseradish peroxidase (1:1000, Jackson ImmunoResearch) for 1h. After washing, blots were developed with ECL Plus Western Blotting detection reagent (Amersham, GE Healthcare) and visualized using a ChemiDox XXRS molecular imager (Bio-Rad).