BioMed Research International / 2010 / Article / Fig 5

Methodology Report

Hunting for Serine 276-Phosphorylated p65

Figure 5

The detected 130 kDa and 80 kDa immunoreactivities are not NF-κB p105/p50 or c-Rel. 1321N1 cells were transiently transfected with control, p105/p50 or c-Rel siRNA as described in Section 2. Cells were induced for 30 min with TNF-α (2000 IU/mL) or iso (10  𝜇 M). Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using Cell Signaling no. 3037 (A1, B1). 130 and 80 kDa immunoreactivities are indicated by arrows (1 and 2 resp.). Because of the overlap of the 130 kDa and the p105 immunoreactive bands, blot A was first stripped and reprobed with anti-p105/p50 (A2). p105 immunoreactivity is indicated by the upper arrow; p50 immunoreactivity is indicated by the lower arrow. Blot B was extensively washed and reprobed (without stripping) with anti-c-Rel (B2). c-Rel immunoreactivity is indicated by an arrow. Tubulin was used as a loading control (A3, B3).