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.