BioMed Research International / 2010 / Article / Fig 4

Methodology Report

Hunting for Serine 276-Phosphorylated p65

Figure 4

Presence of anti-P-p65 Ser276-reactive bands in p65 deficient MEF cells. p65 knock-out MEF cells ( / ), or p65 / MEF cells reconstituted with wild type p65 (p65 wt) or p65 with Ser276 mutated to alanine (p65 S/A), were induced with forskolin (forsk, 10  𝜇 M) for 0, 30, and 60 min. Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using Cell Signaling no. 3037 (A1) or SAB no. 11011 (B1). Blots were extensively washed and reprobed with anti-p65 to confirm p65 absence in / MEFs, and p65 presence in wt and S/A reconstituted MEFs (A2 and B2, arrows indicate anti-p65 detected bands).