Ik2 phosphorylates Spn-F and the phosphorylation is not essential for physical interaction between the proteins. (A) Western blot analysis for mobility shift assay. Lane 1 – S2 cells expressing Spn-F. Lane 2 – S2 cells expressing Spn-F that were treated with calf intestinal alkaline phosphatase (CIP). Lane 3 – S2 cells co-expressing Spn-F and Ik2. Lane 4 – S2 cells co-expressing Spn-F and Ik2 that were treated with CIP. Spn-F co-expressed with Ik2 migrates more slowly on the gel than Spn-F alone or after treatment with CIP. (B) In vitro kinase assay. Autophosphorylation of Ik2 and phosphorylated Spn-F is indicated in lane 2. GFP or GFP-Ik2 was expressed in S2 cells and immunoprecipitated by anti-GFP. The immunoprecipitated proteins were subjected to an in vitro kinase assay using recombinant HIS-Spn-F protein. (C) Western blot analysis for mobility shift assay. Lanes 1, 4 – S2 cells expressing GFP-Spn-F alone. Lane 2 – GFP-Spn-F and kinase dead Ik2 – IK2 [K41A]-Myc. Lane 3 – GFP-Spn-F and Ik2. Kinase dead Ik2 does not affect the mobility of Spn-F whereas Ik2 retards its migration on the gel. (D) Co-immunoprecipitation of Ik2 [K41A]-Myc with HA-Spn-F. Proteins were extracted from S2 cells co-transfected with Ik2 [K41A]-Myc and HA-Spn-F. Untreated lysate was run on gel (Total). The same lysate was incubated with α-myc antibody and precipitated by protein A-coated beads (IP-α-myc), or was precipitated by protein A-coated beads without anti-myc antibody (control). To detect Spn-F, Western blotting was performed using α-HA antibodies. HA-Spn-F was successfully precipitated with kinase dead Ik2.
Dubin-Bar et al. BMC Cell Biology 2008 9:51 doi:10.1186/1471-2121-9-51