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. 2009 Mar 23;184(6):785–792. doi: 10.1083/jcb.200810155

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© 2009 Zhang et al.

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Figure 2. — RIL colocalizes and interacts with Src. (A–F) WI-38 cells were dually stained with anti-RIL mAb 2A2.11 (A and D) and rabbit anti-Src (B) or anti-pY419 Src (E) antibodies. C and F show merged images. (G) 312.5 ng MBP-Src was incubated with GST or GST-RIL as indicated. The input (lane 1; 10 ng/lane), GST (lane 2), and GST-RIL (lane 3) pull-downs were analyzed by Western blotting and Coomassie blue staining. (H) 250 ng MBP-RIL was incubated with GST or GST fusion protein containing Src SH3, SH2, or kinase domain (KD) as indicated. The input (lane 1; 2 ng/lane) and GST or GST fusion protein pull-downs (lanes 2–10) were analyzed by Western blotting and Coomassie blue staining. (I) 250 µg of lysates of HCT116 cells transfected with Flag-RIL or the control vector were mixed with 20 µl of M2-conjugated agarose beads. Lane 1 was loaded with 5 µg of the control cell lysates (to show the position of Src). The immunoprecipitates were analyzed by Western blotting with anti-Src or anti-Flag antibodies. (J) Anti-RIL immunoprecipitates were prepared by mixing 500 µg WI-38 cell lysates with 5 µg anti-RIL mAb. The lysates (lane 1; 5 µg/lane) and anti-RIL immunoprecipitates (lane 3) were analyzed by Western blotting with anti-Src or anti-RIL antibodies. The sample in lane 2 was prepared as in lane 3 except the lysates were omitted (to show the IgG bands derived from the anti-RIL antibody used in immunoprecipitation). Mr, molecular mass; WB, Western blot; IP, immunoprecipitation. Bar, 15 µm.