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. 2016 Dec 27;16(2):213–223. doi: 10.1080/15384101.2016.1261767

Figure 4.

Figure 4.

LZAP enhances the intrinsic catalytic activity of Wip1 toward phosphopeptides and binds MDM2 and HuR. (A-C) Phosphorylated peptides derived from MDM2 (A), ERK1 (B), and HuR (C) were used as substrates for in vitro Wip1 phosphatase assays. Substrates were incubated with the indicated amounts of bacterially-produced His-Wip1 and His-LZAP in phosphatase buffer with or without magnesium for 30 minutes at 30 degrees. Phosphatase reactions were mixed with the malachite green substrate, incubated for 30 minutes at room temperature, and OD620 was measured. Absorbance values were converted to levels of released phosphate using a standard curve. Data presented are the mean of at least 2 independent experiments, and error bars represent standard deviation. * and ** indicate p<0.05 and p<0.01, respectively, as calculated by 2-tailed Student t-test. (D) LZAP and MDM2 interact in mammalian cells. U2OS cells were transfected with indicated plasmids encoding tagged Flag-LZAP or Myc-MDM2, and ectopic expression of these proteins was confirmed by immunoblot of whole cell lysate (bottom panels). Immunoprecipitates were prepared using Myc affinity matrix (top panels) to pull down MDM2 or Flag affinity matrix to pull down LZAP (middle panels), resolved on SDS-PAGE, and immunoblotted by antibodies recognizing Myc(-MDM2) or Flag(-LZAP). (E) LZAP and ERK1 do not interact in mammalian cells. U2OS cells were transfected with indicated plasmids encoding tagged Myc-LZAP, HA-ERK1, or HA-ARF, and ectopic expression of these proteins was confirmed (right panels). Immunoprecipitates were prepared using Myc affinity matrix to pull down LZAP (left panels), resolved on SDS-PAGE, and immunoblotted by antibodies recognizing HA(-ERK1 and ARF) and Myc(-LZAP). (F) LZAP and HuR interact in mammalian cells. U2OS cells were transfected with indicated plasmids encoding tagged Myc-LZAP or Flag-HuR, and ectopic expression of these proteins was confirmed (bottom panels). Immunoprecipitates were prepared using Myc affinity matrix to pull down LZAP (top panels), resolved on SDS-PAGE, and immunoblotted by antibodies recognizing Flag(-HuR) or Myc(-LZAP).