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Journal of Biomedicine and Biotechnology
Volume 2010, Article ID 275892, 9 pages
http://dx.doi.org/10.1155/2010/275892
Methodology Report

Hunting for Serine 276-Phosphorylated p65

Laboratory of Eukaryotic Gene Expression (LEGEST), Department of Physiology, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium

Received 13 August 2009; Revised 11 November 2009; Accepted 17 November 2009

Academic Editor: Susan A. Rotenberg

Copyright © 2010 Anneleen Spooren et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

Supplementary table 1. Summary of reports that have used phospho-specific antibodies to show phosphorylation of p65 at the ser276 residue. In the table the cell types in which p65 phosphorylation was investigated, and the antibody-based techniques used to study p65 phosphorylation, are indicated. All corresponding authors were contacted via E-mail and where available, information (from these personal communications) on the MW of the immunoreactive band shown on anti-P-p65 ser276 Western Blots in the paper is indicated. (NA: not applicable, CS: Cell Signaling, WB: Western Blot, IF: immunofluo, IHC: immunohistochemistry, ChIP: chromatin immunoprecipitation).

Supplementary Figure 1. Tubulin loading controls to Fig. 3. Presence of tubulin in cell lysates was analysed by Western Blot. After anti-p65 + anti-PKAc detection (Fig. 3, A2, B2, C2), blots were reprobed with anti-tubulin to assess equal protein loading.

Supplementary Figure 2. Anti-P-p65 ser276 is not detectable in p65-enriched immunoprecipitates. 1321N1 cells were induced for 30 minutes with vehicle, isoproterenol (iso) or TNF. A. Cell lysates were subjected to immunoprecipitation with beads or anti-p65 (p65 C20). Presence of anti-P-p65 ser276 immunoreactivities in inputs (A1) and immunoprecipitates (B1) was investigated by Western Blotting using Cell Signaling #3037 (A1, B1). Presence of anti-P-p65 ser536 in the same inputs (C1) and immunoprecipitates (D1) was investigated by Western Blotting using anti-P-p65 ser536. Blots B1 and D1 were washed extensively and reprobed with anti-p65 (B2, D2).

IP protocol: Briefly, cells (2 x 106 cells per 10 cm dish) were scraped and lysed in 500 μl RIPA lysis buffer (150 mM NaCl, 50 mM Tris HCl pH 7,5, 1% NP40, 0,01% SDS, 1 mM EDTA, 1 mM EGTA, 0,5% sodium deoxycholate) supplemented with leupeptine, β-glycerophosphate, Aprotinin, Pefabloc, sodium fluoride and sodium orthovanadate. After 1h of rotation, lysates were centrifuged at 14000 rpm for 10 min and supernatant was used for immunoprecipitation. 30 μl of supernatant was isolated before immunoprecipitation and used as input control. After 2 hours of incubation with antibody, 20 μl of A-Beads was added and lysates were rotated overnight. Subsequently beads were washed 3 times with 1 ml of RIPA buffer supplemented with inhibitors. Samples were denatured in 20 μl of RIPA, 25 μl of 2× Laemmli buffer and 2,5 μl of 1M DTT for 10 min at 95°C and used for western blotting as described below.

Supplementary Figure 3. Immunoprecipitation of overexpressed wild type FLAG p65 or FLAG p65 S276A mutant does not lead to recovery of the 130 kDa or 80 kDa immunoreactive bands. 1321N1 cells were induced for 30 minutes with vehicle, isoproterenol (iso) or TNF. A. Cell lysates were subjected to immunoprecipitation with anti-FLAG beads. Presence of anti-P-p65 ser276 immunoreactivities in inputs (bottom panels) and immunoprecipitates (top panels) was investigated by Western Blotting using Cell Signaling #3037. Blots were washed extensively and reprobed with anti-p65. IP protocol was the same as in Suppl. Fig. 3.

  1. Supplementary Material