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. 2012 May 15;122(6):2165–2175. doi: 10.1172/JCI61380

Figure 2. SYK(L) is regulated by CHK1 through phosphorylation on Ser295.

Figure 2

(A) His-tagged SYK(L) WT or the S295A mutant that was translated in vitro were incubated with samples immunoprecipitated from cells with Flag-CHK1 in the presence or the absence of ATP (as indicated) for 2 hours. The samples were then analyzed by Western blot. (B) His-tagged SYK(L) WT protein translated in vitro was incubated with Flag-CHK1 WT or its kinase dead (KD) form, which was immunoprecipitated from cells as indicated for 2 hours. The samples were then analyzed by Western blot. (C) SMMC7721 cells transfected with the indicated plasmids for 24 hours were analyzed by Western blot. (D) SMMC7721 cells were stably transfected with the WT SYK(L) or the mutant SYK(L)-S295A as indicated, and the cells were treated with GÖ6976 (100 nM) or HU (2.5 mM) for 10 hours as indicated and subjected to Western blot. (E) Huh7 cells were transfected with scramble or 3 different CHK1 siRNA duplexes as indicated for 48 hours. The cell lysates were resolved directly by SDS-PAGE (WCL) or were first incubated with anti-SYK(L) antibodies (IP: SYK) and then analyzed by Western blot.