Figure 2

Tyrosine phosphorylation on mitotic spindles. Taxol-stabilized mitotic spindles were isolated from cells released from nocodazole-induced arrest (see “Methods”). (a) Mitotic spindles isolated from parental HeLa cells (upper panels) and HeLa S3/Fyn cells (lower panels) were doubly stained for endogenous Fyn (red) plus α-tubulin (green) (upper panels) and expressed Fyn (red) plus α-tubulin (green) (lower panels). Scale bar, 10 μm. (b) Mitotic spindles isolated from HeLa S3/Fyn cells were doubly stained for Fyn (red) plus α-tubulin (green) (upper panels) and pTyr (red) plus α-tubulin (green) (lower panels). Scale bars, 10 μm. (c–e) Mitotic spindles isolated from (c) HeLa S3/Fyn cells, (d) parental HeLa S3 and HeLa S3/Fyn cells, and (e) HeLa S3/Fyn cells treated with or without 10 µM PP2 were lysed and analyzed by Western blotting using the indicated antibodies. Cellular tyrosine phosphorylation levels are shown relative to that in each control sample following normalization with α-tubulin levels.