Figure 3

DdRACK1 forms homodimers and oligomers and is a phosphotyrosine-containing protein. (A) and (B) Analysis of DdRACK1 dimerization ability. 5–10 μg/100 μl of recombinant DdRACK1 (A) and DdRACK1mut (B) were incubated with 0.001% of the cross-linker glutaraldehyde and samples taken at the indicated time points of 5, 10 and 20 min. For both DdRACK1 and DdRACK1mut, in the absence of glutaraldehyde, the monomers (36 kDa, mono), including dimers (72 kDa), and trimers (108 kDa) were detected. Protein bands which correspond to tetramers were also detected for DdRACK1. Proteins were detected with polyclonal anti-DdRACK1 antibodies. (C) Co-immunoprecipitation analysis using GFP-DdRACK1 and GFP-DdRACK1mut. Both GFP-DdRACK1 and GFP-DdRACK1mut bound to GFP-trap beads (upper panel) were able to immunoprecipitate endogenous RACK1 (lower panel). For GFP-RACK1 fusions, degradation bands were observed. GFP-trap beads incubated with AX2 wild type cell lysate was used as control (Ctl). (D) Detection of DdRACK1 as a phosphotyrosine-containing protein. Western blot analysis was performed with proteins from immunoprecipitated GFP-DdRACK1 cell lysates (upper panel) prepared in presence (+) or absence (−) of phosphatase inhibitor cocktail (PIC). AX2 cell lysate incubated with GFP-trap beads was used as control (Ctl). The phosphotyrosine specific mAb 5E7 detected GFP-DdRACK1 in the IP (lower panel).