BioMed Research International / 2010 / Article / Fig 2

Methodology Report

Hunting for Serine 276-Phosphorylated p65

Figure 2

The Cell Signaling no. 3037 anti-P-p65 Ser276-immunoreactive band persists in cells where p65 is silenced via an siRNA approach. 1321N1 cells were transiently transfected with control or p65 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 Cell Signaling no. 3037 (A1). 130 and 80 kDa immunoreactivities are indicated by arrows. Western Blot to detect anti-P-p65Ser536 (65 kDa arrow) was performed on the same lysates (B1). Blots A1 and B1 were extensively washed and reprobed with anti-p65 to investigate efficiency of p65 knock-down. Anti-p65 detected bands are marked with arrows (A2, B2). Finally, blots were reprobed with anti-tubulin to check loading efficiency. Anti-tubulin-detected bands are marked with arrows (A3, B3).