JCS02418 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 (Adobe PDF) -

  Fig. S1.  bni1 deletion reduces mating MAPK activation. Fus3 kinase activity in the experiments shown in Fig. 2A,B was quantified. Films were scanned and bands were analyzed with Scion Image software.  The entire lane (including bands of both endogenous and exogenous substrates) of individual kinase assays was quantified and normalized to the level of Fus3 protein.  Relative values (wild type before treatment was set to 1) are shown.  (A) Dose response;  (B) time course.  Values represent mean ± s.d. of two independent experiments. 

• Supplemental Figure 2 (Adobe PDF) -

  Fig. S2.  Ste5 recruitment in she mutants. Wild type (WT; EYL1684), she1 (EYL1685), she4 (EYL1688) and she5 (EYL1689) cells expressing Ste5-Myc9 (EBL453) were treated with 5 mM a factor for 0, 15, 45 and 120 minutes. Ste5-Myc was detected by analysis of indirect immunofluorescence as in Fig. 3B. 

• Supplemental Figure 3 (Adobe PDF) -

  Fig. S3. Bni1-GFP and Ste5-Myc9 recruitment in rho2-5 mutants. Wild type (WT), rho2, rho3, rho4 and rho5 null mutant cells expressing Bni1-GFP or Ste5-Myc9 were treated with 5 mM a factor for 2 hours.  Bni1-GFP was visualized by direct fluorescence microscopy and Ste5-Myc9 was visualized by indirect immunofluorescence.

• Supplemental Figure 4 (Adobe PDF) -

  Fig. S4. Ste5-Myc9 recruitment in myo mutants. (A) Ste5-Myc9 localization in myo2-66/myo4 cells.  TAgNLSK128TSte5-Myc9 was expressed in myo2-66 (QMY630), myo2-66/myo4 (QMY632) and myo4 (QMY631) strains.  The cells were treated as in Fig. 7A. (B) Ste5 localization in myo1 mutants.  WT (EY699) and myo1 (EYL1098) cells expressing Ste5-Myc9 (EYL453) were treated with 5 mM a factor for 30 and 60 minutes, then fixed and Ste5-Myc9 was detected by indirect immunofluorescence.  Ste5-Myc9 rim staining was examined in over 300 cells. (C) Ste5-Myc9 recruitment in myo3 and myo5 mutants.  WT (QMY550), myo3D (QMY483), myo5(QMY484), or myo3/myo5 (QMY485) cells expressing Ste5-Myc9 (EBL453) were treated with 5 mM a factor for 60 minutes.

• Supplemental Table (Adobe PDF)