Figure 6. LYN^D189Y exhibits a gain of function compared with LYN^WT.

(A) MCF-7 cells stably transduced with GFP, LYN^WT, or LYN^D189Y were plated in 10% FBS. Cell lysates were harvested and analyzed by immunoblot using the indicated antibodies against phosphorylated SRC substrates. (B) MCF-7 or (C) HCC-1428 and ZR 75-1 cells stably transduced with GFP, LYN^WT, or LYN^D189Y were incubated in serum-free media for 24 or 48 hours, and then protein lysates were prepared and analyzed by immunoblot using the indicated antibodies. (D) For in vitro kinase assays, lysates from cells in A were precipitated with a V5 antibody, as described in the Methods. Kinase reactions were done in the presence or absence of 0.2 mM ATP and then analyzed by immunoblot with pTyr and V5 antibodies. (E) Cells from A were incubated in serum-free media (SFM) or 10% FBS for 48 hours. Lysates were precipitated with a V5 antibody, and pulldowns were analyzed by immunoblot with Y507 P-LYN and V5 antibodies. Densitometry analysis was performed using ImageJ; the ratios of pTyr/V5 bands in D and P-LYN_Y507/V5 bands in E are shown below the blots.