Immunocytochemical and Western blot analysis of native and mutated Sry protein cellular distribution. Cells were transfected with Sryα (top), Sryβ, Sryγ, Sryα R21H, or Sryβ H21R (bottom) and analyzed with microscopy and western blot for cellular distribution. The first (left most) column of images shows the transfected CHO cells visualized with light microscopy, the second with Cy3 (red) detection of the respective Sry (with Cy3 conjugated to the secondary antibody recognizing the Sry specific antibody), third the DAPI staining (blue, recognizing the nucleus) with Cy3 overlay, and the fourth (right most column) the overlay of all columns allowing for identification of the nucleus and cell outline together with Sry expression (red). To the right of the images of the cells are western blots of the nuclear and cytoplasmic fractions from CHO cell lysates. Sryα and Sryγ proteins localize exclusively to the nucleus while Sry β is distributed in both the nucleus and cytoplasm. Chimeric Sry β(H21R) encodes an R at residue 21, as seen in Sryα/γ, rather than H, while Sry α(R21H) proteins contain a H residue at position 21 as see in native Sryβ. The resulting staining patterns demonstrate that an R encoded at position 21 stimulates nuclear localize while a H at this position as see in native Sry β and chimeric Sryα (R21H) results in cytoplasmic accumulation. Western blots of nuclear and cytoplasmic extractions support the results from the immunocytochemical analysis.