Fig. 2

Ras interacts with the SPRY domain of SPSB1. 293 T cells (a, b, c, d, f) were transfected with indicated DNA constructs (0.5 μg/well each). 48 h later, cell lysates were immunoprecipitated (IP) with anti-Ras antibody (b, d, f) or anti-MYC antibody (c) conjugated with protein G beads or anti-FLAG beads (a). Both whole cell lysates and immunoprecipatates were examined for indicated proteins by immunoblotting (IB). 293 T cells (e) were transfected with v-Ha-Ras or FLAG-SPSB1 singly (top two images) or co-transfected with v-Ha-Ras and FLAG-SPSB1 (bottom images) for 48 h. Fixed cells were then immunostained with rabbit anti-FLAG followed by Alexa⁵⁴⁶-conjugated secondary anti-rabbit IgG and/or mouse anti-Ras followed by Alexa⁴⁸⁸-conjugated secondary anti-mouse IgG as indicated. The sub-cellular localization of SPSB1 (red) and v-Ha-Ras (green) was analyzed by confocal microscope (magnification = 60×). Co-localization of merged images appears as yellow. All experiments were repeated three times, with representative results shown. A figure illustration of SPSB1 protein is shown in a