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. 2004 Dec 15;18(24):3117–3130. doi: 10.1101/gad.314904

Figure 4.

Figure 4.

Fuz7 is required for Crk1 T-loop phosphorylation. (A) Requirement of Fuz7 for Crk1-mediated hyperpolarized growth. FB1, UME61 (FB1 Pcrg1:crk1-myc), and UME63 (FB1 Pcrg1:crk1-myc Δfuz7) cells were incubated in CM-arabinose for 6 h. (Right) DAPI staining of the same cell field. Bar, 10 μm. (B) T-loop phosphorylation of Crk1 in vivo is dependent on Fuz7. Cell extracts were prepared from UME61 (FB1 Δcrk1 Pcrg:crk1-myc), UME60 (FB1 Δcrk1 Δfuz7), and UME63 (FB1 Δcrk1 Pcrg1:crk1-myc Δfuz7) cells and analyzed as described in Figure 2. (C) Crk1 and Fuz7 coprecipitate. Cultures of FB1, UME61 (FB1 Δcrk1 Pcrg:crk1-myc), UMS19 (FB1 fuz7-VSV), and UMS20 (FB1 fuz7-VSV Pcrg1:crk1-myc) strains grown in CMA until OD600 of 0.5 were used to prepare whole-cell extracts (WCE). Immuno precipitants obtained with anti-myc and anti-Fuz7 were analyzed by 8% SDS-PAGE, followed by immunoblotting with anti-VSV-peroxidase and anti-myc-peroxidase antibodies.