Figure 5. UNC119a is required for Fyn signaling for the completion of cytokinesis. (A) Co-immunoprecipitation of Fyn with UNC119a. HeLa cell extracts were prepared as described in Figure 3B and immunoprecipitation was performed using UNC-Ab or Fyn-Ab. Western blots were performed with UNC-Ab, Fyn-Ab, phosphor-Src (pSrc)-Ab [pSrc(Y418)], Yes-Ab and Src-Ab. Pre-immune, immunoprecipitation with rabbit preimmune serum; UNC119a and Fyn, immunoprecipitation with respective Ab; Input, loading control as described in Figure 3B. Arrow indicates IgG heavy chain. (B) UNC119a activates Fyn. Left: UNC119a siRNA treatment depletes endogenous UNC119a but has no effect on the amount of SFKs. Western blotting analyses were performed with indicated antibodies. siN.C, extracts from control siRNA-treated cells; siUNC119, extracts from UNC119a siRNA-treated cells. Middle: UNC119a siRNA treatment inhibits the activation of Fyn and Yes. Upper panel: After immunoprecipitation with Fyn-Ab, western blotting was performed with Fyn-Ab and pSrc-Ab. Lower panel: After immunoprecipitation with Yes-Ab, western blotting was performed with Yes-Ab and pSrc-Ab. Pre, immunoprecipitation with rabbit preimmune serum; Input, loading control as above. Right: UNC119a-depletion has a more significant inhibitory effect on Fyn activation than on Yes activation. The images of three independent western blots were analyzed by LAS-4000 (Fuji Film) using Multi Gauge ver.3.1 to quantitate the degree of inhibition. ***p < 0.001; **p < 0.01. (C) Cell cycle-dependent co-localization of Fyn with UNC119a. Asynchronously growing HeLa cells were fixed with paraformaldehyde and double immunostained with monoclonal Fyn-Ab (green) and UNC-Ab (red). Bar, 10 μm. (D) The interaction of Fyn with UNC119a is independent of Rab11a. Left: Rab11 siRNA treatment depletes endogenous Rab11a. Western blots with the Rab11a-Ab and α-tubulin-Ab. siN.C; control siRNA-treated cell extracts. siRab11; Rab11 (a + b) siRNA-treated cells extracts. Right: After immunoprecipitation with UNC-Ab, western blotting was performed with Fyn-Ab, pSrc-Ab and UNC-Ab. Input, loading control as above. (E) Fyn siRNA treatment inhibits cytokinesis. HeLa cells were treated with Fyn siRNA for 72 h and fixed with paraformaldehyde. After permeabilization, the cells were immunostained with α-tubulin Ab and DAPI. The number of bi-nucleated cells was counted. The graphs represent the mean ± SD of three independent experiments. siN.C: bi-nucleated cells, 2.84 ± 0.42%. n = 1,000: experiment 1, 500; experiment 2, 200; experiment 3, 300. siFyn: bi-nucleated cells, 9.44 ± 0.29%. n = 1,100: experiment 1, 400; experiment 2, 400; experiment 3, 300. siN.C; control siRNA-treated cells. siFyn; Fyn siRNA-treated cells. **p < 0.01. (F) Fyn siRNA treatment inhibits the midbody localization of UNC119a. Fyn siRNA-treated cells were fixed with paraformaldehyde and immunostained with UNC-Ab, ERK2-Ab or phosphor-ERK1/2-Ab. Dark arrows (DIC images) and white arrows (fluorescent images) indicate the midbody. UNC119a, ERK and pERK were not found at the midbody, which is indicated by white arrows. Bar, 20 μm. (G) Fyn siRNA treatment inhibits the phosphorylation of ERK. HeLa cells were treated with Fyn siRNA for 72 h and lysed with RIPA buffer. Western blotting experiments were performed with the indicated antibodies.