BioMed Research International / 2010 / Article / Fig 6

Methodology Report

Hunting for Serine 276-Phosphorylated p65

Figure 6

In vitro recognition of the phosphorylated Serine 276 residue of p65 by Cell Signaling no. 3037. Recombinant wt p65-GST or SC mutant p65-GST fusion proteins were in vitro phosphorylated using recombinant MSK-1. 250 ng of the recombinant proteins were spotted on a nitrocellulose membrane and subjected to Western Blotting with Ser276 phospho-specific p65 antibody, either preincubated with 10  πœ‡ g/mL phosphorylated peptide that was used to generate the antibody (upper panel) or not. 1  πœ‡ g of recombinant protein was separated by SDS-PAGE after the in vitro kinase assays performed either in the presence of active MSK-1 and/or 10  πœ‡ M H89 (lower panel) or not.