Fig 3. Ste5-Myc9 migrates in polyacrylamide gels as full-length, high molecular weight, aggregated and truncated forms of varying proportions in ste5D, ste5D ste11D, cdc28-4 and SSA1-GFP^OP strains.

A. SDS-PAGE of whole cell extracts of Ste5-Myc9 (2m) in bar1D ste5D (EY1775) treated without and with a factor, using a modified H-buffer for extract preparation that has many protease and phosphatase inhibitors and no added NaCl. B. Native PAGE of Ste5-Myc9 (2m) in bar1D ste5D (EY1775) and bar1D ste5D ste11D without and with a factor. The cell extraction buffer is the same as in A but approximately twice as much extract was loaded on the gel. C. The cdc28-4 mutation reduces high molecular weight and aggregated forms of Ste5-Myc9. Ste5-Myc9 was expressed from 2μ (pSKM19) and CEN (pSKM12) plasmids in W303a CDC28 (EY957) and cdc28-4 (PY1236) strains grown at room temperature and after 3 hours at 37°C and whole cell extracts prepared by standard procedures. Lanes 1,2: cdc28-4 STE5-M9-2μ RT, 37°C. Lanes 3, 4: WT STE5-MYC9-2μ RT, 37°C. Lanes 5,6: cdc28-4 STE5-MYC9-CEN RT, 37°C. Lanes 7,8: WT STE5-MYC9-CEN RT, 37°C. D-E. Ste5-Myc9 with excess Ssa1-GFP. The full SDS-PAGE gel of whole cell extracts used for co-immunoprecipitation in Fig 2 are shown at short (D) and long (E) exposure times. F-G. The immunoblot in D. was stripped and reprobed for Ssa1-GFP and GFP-Cdc24. with anti-GFP antibodies, F is a short exposure and G is a long exposure. Labels:] aggregated in loading well, |high mw forms, a] full-length,] b-g degraded fragments. The extraction buffers in C-E were as in Elion et al, 1993 [20] with 200 mM NaCl.