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Figure 6 | BMC Biochemistry

Figure 6

From: The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin

Figure 6

Analysis of a co-immunoprecipitation assay by immunoblotting (A and B) and by phosphorylation assay (C). A: Western Blot analysis using anti-Stk33 for the detection of precipitated Stk33. Lane 1–3: samples of washing steps 1–3; lane 4: immunoprecipitate; the arrowheads point towards IgG contamination. Arrow = Stk33. B: Western Blot analysis using anti-vimentin for the detection of co-precipitated vimentin. Lane 1–3: samples of washing steps 1–3; lane 4: immunoprecipitate; Arrow = co-precipitated vimentin. C: Phosphorylation assay proofing the enzymatic activity of immunoprecipitated Stk33. Electrophoretic separation of the precipitate after in vitro incubation with radiolabeled γ 32P ATP and autoradiography of the gel. An aliquot of the precipitate was incubated with recombinant vimentin ΔN50 (lane 1) and with recombinant vimentin ΔH (lane 2). Arrowheads indicate phosphorylated Stk33 and/or vimentin as a discrimination is not possible due to similar molecular weight of the proteins. The radioactively labelled protein band (arrow) corresponds to phosphorylated vimentin ΔN50 according to co-electrophoresed molecular standard. * indicates an additional protein that co-precipitated by anti-Stk33. The phosphorylation assay was done two times.

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