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. 2010 Nov 17;25(1):138–156. doi: 10.1210/me.2010-0217

Figure 6.

Figure 6

cAMP-independent transactivation of cKIT in NDP-MSH-stimulated HBL cells. A, Kinetics of cKIT activation by NDP-MSH. Serum-starved HBL cells were stimulated with NDP-MSH for the times shown and analyzed for cKIT phosphorylation at Tyr721 by Western blot. B, Lack of cKIT activation by FSK. Cells were treated with FSK (10−5 m, 30 min) or NDP-MSH (10−7 m, 4 min) as positive control and blotted for cKIT phosphorylation at Tyr721 (upper panel), ERK activation (middle panel), or total ERK (lower panel, loading control). C, The cKIT inhibitor ISCK03 blocks ERK activation by NDP-MSH. Cells were transiently transfected with the empty vector (pcDNA3) or with a constitutively active NRAS mutant (Q61R), preincubated with ISCK03 (1 μM, 1 h), and then challenged with NDP-MSH for 4 min. Cell extracts were analyzed by Western blot for pERK. D, Impaired ERK activation by NDP-MSH in HBL cells treated with the cKIT inhibitors GTP-14564 and Sunitinib. HBL cells were pretreated for 1 h with the indicated concentrations of the cKIT inhibitors and then challenged with NDP-MSH (10−7 m, 2 or 4 min) before estimation of pERK levels by Western blot.