Fig. 7

Co-immunoprecipitation and Western Blot analyses of SSG-1 interacting proteins. a corresponds to the results of the Co-IP of SSG-1 and SsSOD, b corresponds to the results of the Co-IP of SSG-1 and SsNramp, c corresponds to the results of the Co-IP of SSG-1 and SsSit and d corresponds to the results of the Co-IP of SSG-1 and SsGAPDH. Whole cell free extracts of S. cerevisiae cells expressing the complete c-myc tagged SSG-1 coding sequence fused to the GAL4 activation domain (prey protein) and the HA tagged protein fragment fused to the GAL4 DNA binding domain (bait protein) were co-immunoprecipitated as described in Methods. The coimmunoprecipitated proteins were separated using 10% SDS polyacrylamide electrophoresis under non-reducing conditions and transferred to nitrocellulose. Lane 1: Nitrocellulose strips were probed with anti-cMyc antibody as the primary antibody and anti-mouse IgG as the secondary antibody that recognizes both the heavy and light chain of the anti-cMyc (The two high molecular weight bands present belong to the anti-cMyc antibody used for precipitation). Lane 2: Negative controls where no primary antibody was added and probed with the secondary antibody anti-mouse IgG that recognizes both the heavy and light chain of the anti-cMyc antibody. Lane 3: Nitrocellulose strips were probed with anti-HA antibody as the primary antibody and anti-rabbit IgG as the secondary antibody that recognizes both the heavy and light chain of the anti-HA. Lane 4: Negative controls where no primary antibody was added and probed with the secondary antibody anti-rabbit IgG that recognizes both the heavy and light chain of the anti-HA antibody. Pre-stained molecular weight markers were included in outside lanes of the gel. Exposure times to detect the interacting proteins SSSOD using anti-cMyc antibodies and antiHA antibodies were 3.1 s and 9.1 s, respectively. To detect the other binding partners SSNRAMP, SSSIT, and SSGAPDH using anti-cMyc antibody and anti-HA antibody were 1 s for each one