Figure 2

Subcellular localization, and developmental level of expression of DdRACK1. (A) To determine the localization of DdRACK1, AX2 wild type cells co-expressing GFP-Gβ and RFP-DdRACK1 were used to perform confocal live cell microscopy. (B) Immunofluorescence studies of AX2 wild type cells. Vegetative cells were fixed and stained with anti-DdRACK1 antibodies. mAb act1-7 against actin was used to visualize the cell cortex. Scale bar, 5 μm. (C) Subcellular fractionation of AX2 and AX2 expressing GFP-DdRACK1 and GFP-RACK1mut after lysis by passing through Nucleopore filters. Protein aliquots separated by SDS PAGE were used to perform western blot analysis. WL, whole cell lysate; L, supernatant from cell lysate (400 × g); S1, P1 (10,000 × g); S2, P2 (100,000 × g). S, supernatant; P, pellet. DdRACK1 and relative amount of GFP-DdRACK1 and GFP-DdRACK1mut were detected in supernatant as well as in pellet samples with polyclonal anti-DdRACK1 antibodies. mAb 47-16-8 detected the cytosolic marker protein α-actinin which served as control. The α-actinin blot for AX2 is shown. (D) DdRACK1 expression levels during development. Western blot analysis was performed with AX2 wild type cell samples collected during starvation in shaking suspension at indicated time points. DdRACK1 was detected with polyclonal anti-DdRACK1 antibodies. For loading control the blot was probed with mAb 188-19-95 which detects cap32.