Figure 1

PAG down-regulation enhances Src kinase activity and proximal signaling. (A) Jurkat T cells were transfected with either non-targeting shRNA (ctrl) or PAGshRNA (PAGsh) and three days later lysed. The top panel shows the PAG downregulation. The Src family kinases Fyn (middle) and Lck (bottom) were immunoprecipitated and in vitro kinase assay (IVK) performed. Phosphorylation was visualized by autoradiography, the amount of precipitated kinase was determined by immunoblotting. Quantification of the kinase bands can be found in Additional file 2: Figure S2. (B) The cells from (A) were stimulated with CD3 and CD28 antibodies as indicated and lysates immunoblotted with specific antibodies. Actin staining is shown as a loading control. Quantification can be found in the supplemental figure. (C) Jurkat T cells were transfected with either control or PAG shRNA and co-transfected with either PAG-YFP, which differs by two nucleotides within the targeting region, or YFP alone. The YFP-tag was included to allow us to distinguish between the over-expressed protein and endogenous PAG. The figure shows that PAG is suppressed, while the expression of PAG-YFP (the resistant molecule) is unaffected. Actin was included as a loading control. The cells were then either left untreated or stimulated for 5 minutes with anti-CD3 (C305). Cell lysates were immunoblotted to analyze proximal signaling as indicated. Actin staining was included as a loading control. Quantification can be found in the supplemental figure. (D) Jurkat T cells were prepared as described in (A) and fractions 2–4 from sucrose density gradients pooled, separated by SDS-PAGE, blotted, and probed as indicated. Each panel is representative of at least three independent experiments. The phosphotyrosine blot in (B) was originally published in Blood: Smida, et al. 2007 [15]. Quantification can be found in the supplemental material (Additional file 2: Figure S2).