Figure 2

Phosphorylation assay. A: Silver stained gel after electrophoretic separation of wildtype (wt) vimentin and different deletion variants of vimentin used in the kinase assay as substrates. Lane 1: wildtype, lane 2: Δ12, lane 3: Δ20, lane 4: Δ30, lane 5: Δ42, lane 6: Δ50.B: Coomassie stained gel of crosslinked vimentin wildtype monomers by using increasing concentration of glutaraldehyde (GA). For practical reasons (see text) crosslinked vimentin tetramers had to be used in the kinase assay. Lane 1: vim wt without GA, lane 2: vim wt plus 0.005% GA, lane 3: vim wt plus 0.01% GA, lane 4: vim wt plus 0.02% GA, lane 5: vim wt plus 0.04% GA, lane 6: vim wt plus 0.06% GA. C: Electrophoretic separation of the products of different kinase assay with various reactions partners after in vitro incubation with radiolabeled γ ³²P ATP and autoradiography of the gel. Lane 1: Only Stk33δ (deletion derivative) tested for autophosphorylation, lane 2: Stk33δ plus casein as substrate, lane 3: Stk33δ plus vimentin wildtype, lane 4: Stk33 (complete kinase domain) plus casein, lane 6: only Stk33 tested for autophosphorylation, lane 8: Protein kinase A (PKA) plus casein as substrate, lane 9: only PKA tested for autophosphorylation, lane 10: casein + γ ³²P ATP only. Lanes 5 and 7 are devoid of samples. D: Electrophoretic separation of Stk33 and vimentin/vimentin deletion derivatives after in vitro incubation with radiolabeled γ ³²P ATP and autoradiography of the gel. Lane 1: Stk33 plus ΔH crosslinked, lane 3: Stk33 autophosphorylation, lane 5: Stk33 plus vimentin wildtype tetramer, lane 6: vimentin wildtype tetramer plus γ ³²P ATP only, lane 7: Stk33 plus vimentin monomer, lane 9: PKA plus vimentin monomer, lane 10: PKA autophosphorylation. Lanes 2, 4 and 8 are devoid of samples. E: Electrophoretic separation of Stk33 and vimentin/vimentin deletion derivatives after in vitro incubation with radiolabeled γ ³²P ATP and autoradiography of the gel. Lane 1: Stk33 plus vim Δ12, lane 3: Stk33 plus vim Δ20, lane 5: Stk33 plus vim Δ30, lane 7: Stk33 plus vim Δ42, lane 9: Stk33 plus vim Δ50, lane 10: Stk33 plus vim ΔH, lane 11: Stk33 plus vim wt, lane 12: Stk33 autophosphorylation. Lanes 2, 4, 6 and 8 are devoid of samples. Thin arrows indicate vimentin/vimentin deletion derivatives as substrate, thick black arrows indicate casein as substrate, black arrowheads indicate Stk33, white arrowheads indicate Stk33δ. To assure the results presented all experiments were carried out at least two times. Some of the assays were iterated up to four times as a positive control.