Interactions of Rlk with Src Family kinases. A, Structure of endogenous Rlk protein (top) and Rlk-GST fusion constructs (bottom). Numbers of the constructs correspond to the lanes in part B. B,The amino-terminus of Rlk can bind the SH3 domains of Src family kinases, Fyn, Lyn and Lck. GST fusion proteins encoding the amino-terminus or the amino-terminus and SH3 domain were separated on a 10% SDS-PAGE, transferred to nitrocellulose, then probed with biotinylated GST-fusion proteins encoding the SH3 domains of various signaling proteins. Lane 1 GST-Rlk amino-terminus, Lane 2 GST-Rlk amino-termus-SH3 domain, Lane 3 GST control. SH3 domain fusion proteins used to probe blots are indicated above each panel. C, Co-immunopreciptation of Rlk and Fyn. 293T cells were transfected with expression constructs of Rlk or FynT alone or in combination, and lysed at 24 hours. Equal amounts of detergent-soluble cell lysates were immunoprecipitated with anti-Rlk; immune complexes were washed, resolved by SDS-PAGE and immunoblotted (IB) with anti-Fyn-3 (Santa Cruz) (top panel), then stripped and blotted with anti-Rlk (bottom panel). Fyn expression was assessed by immunoblotting equivalent amounts of cell lysate with anti-Fyn (middle panel). Lane 1: Mock transfection, Lane 2: Rlk WT, Lane 3: Fyn WT, Lane 4: Rlk +Fyn.