Figure 2.

E202A expression leads to decreased serine/threonine phosphorylation in post-nuclear supernatants. (A) Post nuclear lysates (20 μg total protein per lane) prepared from WIF-B cells expressing wild type (WT) or the kinase dead STK16 (E202A) were immunoblotted for STK16 (with anti-V5 antibodies) (top panel) or α-tubulin (as a loading control). The asterisk marks a known degradative species of the mutant kinase. Molecular weight standards are indicated on the left (in kDa). (B) The same lysates as in A were immunoblotted (IB) with antibodies against phosphorylated-serine/threonine residues (p-ser/thr). Molecular weight standards are indicated on the left (in kDa), arrows and brackets on the right indicate proteins with decreased immunoreactivity in E202A expressing cells. (C) Post-nuclear lysates were prepared from WIF-B cells expressing wild type (WT) or E202A. 550 μg of total protein per gel were separated by 2D electrophoresis and immunoblotted with anti-phospho-serine/threonine antibodies. The pH gradient of the first dimension is indicated across the top and the molecular weight standards are indicated on the left in kDa. The Coomassie blue stained gels (CBB) are shown in the upper panels and the corresponding immunoblots (IB) are shown below. Samples were processed in duplicate (technical replicates). A representative pair from each condition is shown. Arrowheads mark three examples of proteins with decreased immunoreactivity in E202A expressing cells.