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. Author manuscript; available in PMC: 2013 Apr 24.
Published in final edited form as: Nat Chem Biol. 2007 Jan 28;3(3):156–160. doi: 10.1038/nchembio859

Figure 2.

Figure 2

fmk-BODIPY irreversibly targets RSK but has only modest selectivity and potency in cells. (a) Chemical structure of fmk-BODIPY, a fluorescent fmk derivative. (b) Covalent labeling of RSK2 CTD by fmk-BODIPY. RSK2 CTD was treated with the indicated concentrations of unlabeled fmk for 1 h and then with 3 μM fmk-BODIPY for 1 h. Proteins were resolved by 10% SDS-PAGE and detected by in-gel fluorescence scanning, followed by western blot with an antibody to RSK2. (c) Effect of fmk-BODIPY on PMA-stimulated RSK Ser386 phosphorylation. HEK 293 cells were deprived of serum for 2 h and then treated with the indicated concentrations of fmk-BODIPY for 1 h. Cells were stimulated with PMA (0.1 μg ml−1) for 30 min and harvested in PBS. Proteins were resolved by 10% SDS-PAGE and detected by western blot with antibodies to phospho-Ser386 RSK (pS386) and total RSK2. (d) Selectivity of protein modification by fmk-BODIPY in intact cells. Proteins in cell lysates from c were resolved by 10% SDS-PAGE and detected by in-gel fluorescence scanning (left) and Coomassie blue staining (right). The asterisk marks the position of RSK1 and RSK2.