Mouse monoclonal anti phospho iκbα ser32 36

Formalin-fixed, paraffin-embedded tissue sections were stained with rat monoclonal anti-KI67 antibody (Clone TEC3, DakoCytomation, Carpinteria, CA) diluted 1:25, or mouse monoclonal anti-phospho-IκBα-Ser32/36 (9246, Cell Signaling, Danvers, MA) diluted 1:100 using the Rat Vector Elite ABC Kit (Vector Laboratories, Burlingame, CA) or the Mouse on Mouse (M.O.M) Peroxidase Kit (Vector Laboratories, Burlingame, CA), following the manufacturer´s protocol. Quantitation of staining results was perfomed using ImageJ software to count positive cells.

The differentiated macrophages were treated with IMQ (10 μg/mL), Poly(I:C) (20 μg/mL), or LPS (1 μg/mL) for 30 min, then cells were lysed in Radioimmunoprecipitation (RIPA) buffer (50 mM Tris‐HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP‐40, 0.5% SDS, 1 mM Na3VO4, and Complete Protease Inhibitor Cocktail (Roche Diagnostic, Indianapolis, IN). Total proteins were measured by DC TM Protein Assay, according to manufacturer's instructions (Bio‐Rad Laboratories, Inc., Berkeley, CA).
Western blot analysis for Phospho‐Iκ‐Bα (mouse monoclonal anti‐Phospho‐Iκ‐Bα (Ser32/36), 1:1000; Cell Signaling Technology) was performed after loading 5 μg of cell lysate/lane on NuPAGE 4–12% Bis‐Tris Protein Gels (Thermo Fisher Scientific, Waltham, MA). After the anaylsis of Phospho‐Iκ‐Bα, the membrane was stripped and blotted with mouse monoclonal anti‐Iκ‐Bα (L35A5) (1:1000; Cell Signaling Technology). Optical density values were internally normalized using mouse monoclonal anti‐Vinculin (1:2000; Sigma‐Aldrich) and further corrected for the value of controls considered equal to 1.