Anti phospho iκbα antibody

Immunoprecpitated samples were separated by SDS-PAGE (4–12%) and transferred to nitrocellulose membranes (GE Helathcare). Membranes were incubated with Milk (5%, Tris Buffered Saline Tween, TBST) and with an anti-IκBα antibody (1:1000; C21 rabbit polyclonal, O/N, 4°C, Santa Cruz), an anti-phospho-IκBα antibody (1/1000, Cell Signalling), an anti-spectrin antibody (1/1000) and with anti-β-actin (1/1000), washed in 1x TBST, incubated with IRDye 800CW Donkey anti-Rabbit (1:10,000, 1 hr; LI-COR Biosciences), washed, and developed using LI-COR Odyssey. The intensity of a 135kDa band, constituting the 80kDa spectrin and the IκBα-EGFP, was determined using ImageJ (Version 1.66f or J64), as previously [13 (link),14 (link)].
Pre-immunoprecipitation samples (input) were analysed as above and input levels of spectrin, actin, IκBα and IκBα phosphorylation determined for all conditions. Additional controls included samples immunoprecipitated with a non-specific IgG, which showed no signal.

Retinas were harvested immediately after animals were euthanized and homogenized in a lysis buffer (50mM Tris–HCl (pH=7.5), 150mM NaCl, 5mM EDTA, 1% (v/v) NP-40, 5% (v/v) glycerol, and protease inhibitor cocktail (Calbiochem, San Diego, CA). Protein content was measured using BCA protein assay (Pierce, Rutherford, IL). Total protein from each sample (15μg) was electrophoresed on 4–20% gradient SDS polyacrylamide gels (Invitrogen, Carlsbad, CA), and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were incubated in blocking buffer [5% BSA in TBST (20mMTris–HCl, pH 7.5, 100mMNaCl, and 0.1% Tween-20)] for 1 h at room temperature, and then incubated overnight at 4°C with rabbit polyclonal anti-phospho-IκBα antibody (#2859, Cell Signaling Technology, Beverly, MA), in blocking buffer, followed by 1-hour incubation at room temperature with HRP-conjugated goat anti-rabbit secondary antibody (Amersham Biosciences, Piscataway, NJ). Signals were visualized using Super Signal West Dura extended duration substrate (Pierce, Rutherford, IL) and quantified by conventional digital image analysis using an Image Station 4000R (Eastman Kodak, Rochester, NY). Blots were stripped and reprobed with rabbit polyclonal anti-β actin (ab6276, Abcam) followed by appropriate secondary antibodies for quantification of bands.