FIGURE 5.
Role of the Pro-rich C terminus of SK1 in regulation by PP2Ac. A, comparison of the SK1 and SK1ΔCT sequences, indicating the Pro-rich C-terminal region that is not present in SK1ΔCT. B, HEK293 cells were transfected with vectors encoding either FLAG-tagged SK1 or SK1ΔCT, with either vector encoding HA-tagged PP2Ac or the corresponding empty vector (EV). Cell lysates were immunoprecipitated with anti-FLAG antibody to capture SK1/SK1ΔCT and the presence of associated PP2Ac was detected by immunoblotting with anti-HA antibody (upper panel). The bracket in the lower panel indicates the presence of equal amounts of immunoprecipitated SK1/SK1ΔCT. C, HEK293 cells were transfected as in B, and cell lysates were immunoblotted with an antibody recognizing phospho-SK1 (upper panel). Total SK1/SK1ΔCT and PP2Ac expression levels were confirmed by immunoblotting with anti-FLAG and anti-HA antibodies, respectively (lower panel). Results are representative of three independent experiments. The dividing lines indicate where lanes have been spliced to simplify viewing, but results in each panel are from a single experiment performed on the same day. Detected immunoglobulin heavy chain (IgGH) from the immunoprecipitating antibody is indicated.