BioMed Research International / 2010 / Article / Fig 1

Methodology Report

Hunting for Serine 276-Phosphorylated p65

Figure 1

Anti-phospho-p65 Ser276 (Cell Signaling no. 3037) detects atypical (with MWs of 80 and 130 kDa), inducible immunoreactivities in different cell types, induced with different NF-κB-activating agents. Cells were stimulated for increasing times (L929sA, A549, L363) or for 30 min (Raw264.7, C2C12, 1321N1) with TNF-α (2000 IU/mL), LPS (1  𝜇 g/mL), PMA (10 ng/mL), forskolin (forsk, 10  𝜇 M) or isoproterenol (iso, 10  𝜇 M). For blocking experiments, lysates were loaded on one gel in duplicate and after transfer, blots were cut in half. Immunodetection was performed in parallel, using Cell Signaling no. 3037 or Cell Signaling no. 3037 preincubated for at least 30 minutes with double volume of blocking peptide (BLOCK). Arrow 1 indicates an immunoreactive band with an MW of 130 kDa; arrow 2 indicates an immunoreactive band of 80 kDa.