Anti actin hrp antibody

Cells treated with various drug combinations were washed with cold PBS and lysed in RIPA buffer containing 1mg ml⁻¹ DNAse (Roche Life Science, Indianapolis, IN) and supplemented with protease inhibitor cocktail (Roche Life Science, Indianapolis, IN). Cell lysates, stored frozen at −80 °C were resolved on 4–20% SDS-PAGE gels and transferred onto a nitrocellulose membrane. The membranes were first stained with anti-BIM (Cell Signaling Technology, Danvers, MA), and anti-HOXD10 (Santa Cruz Biotechnology, Dallas, TX) antibody cocktail followed by peroxidase conjugated-anti-rabbit antibody. ECL (Roche Life Science, Indianapolis, IN) was used to develop blots. Signal intensity was recorded on an IVIS Spectrum imaging station (Perkin Elmer, Hopkinton, MA). After documenting signal, the blot was subjected to a second round of immunostaining using an anti-actin-HRP antibody (Santa Cruz Biotechnology, Dallas, TX).

Immunoblotting was performed as described previously (16 (link),18 (link)). Briefly, cells were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 10 mM Na₂HPO₄, pH 7.4) plus protease inhibitor cocktail for 20 min and cleared for insoluble debris by centrifugation at 15,000 × g for 15 minutes at 4°C. Protein concentrations were standardized using DC Protein Assay (Bio-Rad, Hercules, CA) and an aliquot of the lysate was denatured in 2X SDS-sample buffer and boiled for 5 minutes. Equivalent concentrations of lysates were resolved by SDS–PAGE, followed by transfer to PVDF membranes, and immunoblotted with the appropriate antibody. Blots were visualized with HRP-conjugated secondary antibody followed by ECL. Blots were stripped by incubation in glycine IgG stripping buffer for 30–45 minutes with agitation (25 mM Glycine, 1% SDS, 50°C, pH 2.0) for re-probing. Polyclonal anti-COX-2 antibody (160126) was purchased from Cayman Chemical (Ann Arbor, MI), rabbit anti-FFA4 antibody was purchased from Novus Biologicals (Littleton, CO), rabbit anti-phosphoThr202/Tyr204-p44/p42 MAPK (ERK1/2) and p44/42 MAPK (ERK1/2) antibodies were purchased from Cell Signaling Technologies (Beverly, MA), and anti-actin-HRP antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).