Smash is a phosphorylation target of Src42A. (A) The indicated constructs were transfected into S2 cells. Lysates were either directly analyzed by Western blot (Input) or subjected to IP with anti-GFP, followed by Western blot with the indicated antibodies. HA, hemagglutinin epitope tag. (B) Endogenous Smash is tyrosine phosphorylated in vivo. Protein lysates of w¹¹¹⁸ (control) or smash³⁵ mutant embryos were directly analyzed by Western blot (Input) or subjected to IP with anti-Smash intra, followed by Western blot with the indicated antibodies. Note that in the IP a single band of ∼220 kD, which is absent in smash³⁵ mutant embryos, is detected by anti-PY. This band corresponds in size to the band detected by anti-Smash N-term. The band of 80 kD visible in the input blot with anti-Smash N-term in both WT and smash³⁵ mutant lysates is unspecific. (C) The indicated constructs were transfected into S2 cells. Lysates were subjected to IP with anti-GFP, and Western blots were probed with anti-PY and anti-GFP.