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

Figure 3.

Figure 3

OS mutants of GFP-Mid1 associate with FLAG-α4. (a) COS7 cells were transfected to express GFP-Mid1(1–480) together with each of the following FLAG constructs: FLAG empty vector, FLAG-α4, and FLAG-α4(220–340). Cells were fixed and GFP-Mid1 was visualized by direct fluorescence microscopy. FLAG-α4 was visualized by anti-α4 immunofluorescence. Nuclei of the cells were visualized by DAPI staining. (b) COS7 cells were cotransfected to express FLAG-α4 and different GFP-Mid1 fusion constructs: GFP alone (as control), GFP-Mid1, GFP-Mid1(1–480), GFP-Mid1(L626P), and GFP-Mid1(C266R). Immunoprecipitation was performed by using anti-FLAG M2 affinity gel and FLAG-α4; GFP-Mid1 fusion proteins and PP2A were detected by immunoblotting with anti-FLAG, anti-GFP, and anti-PP2A. The mutant GFP-Mid1 proteins all bound FLAG-α4.