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. 2001 May 22;98(12):6650–6655. doi: 10.1073/pnas.111154698

Figure 1.

Figure 1

Association of α4 with Mid1 and PP2A. (a) Yeast two-hybrid analysis showed that strains expressing Gal4-DBD-α4 grew on triple-drop out media (trp-, leu-, his-) only when also expressing PP2A, PP6, or Mid1 (residues 58–180), indicative of an interaction with α4. Controls included single transformants and PP1C that did not support growth, because it does not bind α4. The plate was scanned on a densitometer to show growth of yeast colonies. (b) COS7 cells were transfected with FLAG-tagged Mid1(58–180) with or without myc-tagged α4. Cells were lysed and immunoprecipitation was performed by using anti-FLAG M2 affinity gel. FLAG-Mid1(58–180) and α4 were detected in the immunoprecipitates by immunoblotting with anti-FLAG and anti-α4 antibodies. When present, myc-α4 was detected by anti-myc immunoblotting. (c) COS7 cells transfected with vector alone, FLAG-α4 (full-length), FLAG-α4(1–249), FLAG-α4(220–340), or FLAG-α4(1–111) were lysed, and anti-FLAG immunoprecipitation was performed. FLAG-α4 coprecipitated endogenous Mid1 and PP2A-C, detected by anti-FLAG, anti-Mid1, and anti-PP2A-C immunoblotting.