Fig. 1. Disruption of the svkA gene encoding severin kinase. (A) Two fragments of the kinase were amplified by PCR from genomic DNA, ligated to the blasticidin-resistance cassette and electroporated into AX2 wild-type cells. (B) Blasticidin-resistant clones were tested for knockout events by PCR1 (primers 1 and 2, 1385 bp for knockout) and PCR2 (primers 1 and 3, 2038 bp as a wild-type control). Under the conditions used, PCR2 did not ever amplify 3400 bp, including the blasticidin-resistance cassette, in the knockout, but the lack of a PCR product indicated a disruption of the svkA gene as well. (C) Western blot of 2x10⁵ cells per lane, visualized with kinase-specific antibodies confirms the knockout at the level of SvkA protein and also shows the expression of the GFP fusion protein in the wild-type (first lane) and the mutant background (fourth lane). (D) GFP-SvkA-FL, immunoprecipitated from rescue cells, phosphorylated domains 2 and 3 of severin (DS211C, left), myelin basic protein (MyBP, right) and itself (both lanes).