Rabbit anti iκb

Cell lysates were collected using Protease arrest (GBiosciences, Maryland Heights, MO) in cell lysis buffer (Cell Signaling Technology, Danvers, MA). Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermofisher, Waltham, MA). 4–20% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio–Rad, Hercules, CA) were loaded with 30 μg protein, electrophoresed and transferred to nitrocellulose (Pall Corporation, Ann Arbor, MI), and membranes were blocked for 60 minutes in Blocker casein in tris-buffered saline (TBS) (from Thermofisher, Waltham, MA). Blots were incubated with primary antibodies overnight in block buffer + Tween20 (1:1000) with rabbit anti-IκB and rabbit anti-actin (1:1000, antibodies all purchased from Cell Signaling, Danvers, MA). Blots were washed six times for 5 minutes each in TBS-Tween-20, followed by incubation with secondary antibody (1:10,000) for 60 minutes at room temperature (Cell Signaling, Danvers, MA). Size estimates for proteins were obtained using molecular weight standards from Bio–Rad (Hercules, CA). Blots were visualized and quantified using a LiCor Odyssey CLx Infrared imaging system (Lincoln, NE). After background subtraction, fluorescence intensity for the protein of interest was normalized to the signal intensity for the actin calculated relative to unactivated samples, using Image Studio 4.0 (LiCor).