BioMed Research International / 2010 / Article / Fig 3

Methodology Report

Hunting for Serine 276-Phosphorylated p65

Figure 3

Four independent anti-P-p65 Ser276 antibodies detect inducible bands, with MWs of 80 and 130 kDa, that do not disappear upon p65 knock-down, but are inhibited when PKAcα is silenced. 1321N1 cells were transiently transfected with control, p65 or PKAcα siRNA, as described in Section 2. Cells were induced for 90 min with TNF-α (2000 IU/mL), iso (10  𝜇 M) or a combination of both stimuli. Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using SAB no. 11011 (A1), Rockland no. 100-401-264 (B1), Cell Signaling no. 3037 (B3), or a homemade anti-P-p65 Ser276 antibody (C1). 130 and 80 kDa immunoreactivities are indicated by arrows (1 and 2, resp.). Blots were extensively washed and reprobed with a mixture of anti-p65 and anti-PKAcα to investigate knock-down efficiency. Anti-p65-detected bands are marked by the upper arrow, anti-PKAcα-reactive bands are marked by the lower arrow (A2, B2, C2). Tubulin loading controls are added as Supplementary Figure  1.