FIGURE 1:

Dazl is a novel substrate of MK2. (A) Reduction of MK2 expression by shRNA. Cell lysates from SUM149 cells stably expressing either control or MK2 shRNA were resolved by SDS–PAGE (on two separate gels) and immunoblotted with the indicated antibodies. (B) Schematic of the screen used to identify novel MK2 substrates. Lysates from control and MK2 shRNA cells were incubated with human protein microarrays and probed with anti-phosphoserine and anti-phosphothreonine antibodies, followed by detection with Alexa 488–conjugated secondary antibodies. Arrays were scanned and quantified. See Materials and Methods for additional details. (C) Bar graph of the top 20 putative MK2 substrates identified from the screen. Data are plotted as the average fold signal reduction obtained from the MK2- knockdown shRNA array compared with the control shRNA knockdown array. Error bars indicate SD. (D) In vitro kinase assay using GST-Dazl and MK2. GST-Dazl (or no substrate) was incubated in the presence or absence of active recombinant MK2 and ATPγS, followed by treatment with PNBM. Kinase reactions were resolved by SDS–PAGE (on three separate gels), followed by immunoblotting with the indicated antibodies.