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. Author manuscript; available in PMC: 2018 May 1.
Published in final edited form as: Neuropharmacology. 2017 Mar 1;117:387–400. doi: 10.1016/j.neuropharm.2017.02.027

Fig. 4. Plk2 interacts directly with APP.

Fig. 4

(a) COS-7 cells were transfected with myc-tagged Plk2-KD and either myc-tagged APP or empty vector. Lysates were immunoprecipitated with APP 6E10 antibody or IgG and immunoblotted with 6E10 and Plk2 H-90 antibodies as indicated. (b) Rat cortical neurons (20 DIV) were infected with Sindbis-Plk2-KD. Lysates were immunoprecipitated with Plk2 7382 antibody or IgG and immunoblotted with Plk2 7382 and APP N-terminal antibodies. Overexposure (bottom) is required to visualize endogenous full-length APP in input lane. Each blot was obtained from same gel with same exposure and spliced together for concise display. (c) Rat cultured hippocampal neurons (24 DIV) were harvested and immunoprecipitated with Plk2 antibody (H-90), APP antibody (Y188), or nonimmune rabbit IgG and immunoblotted with H-90 and Y188 to detect endogenous APP and Plk2 proteins. (d) APP-SwDI mouse (4-month-old) forebrain lysates were incubated with IgG or Plk2 antibody N-17 or H-90 and immunoblotted with Plk2 N-17 and human/rodent APP C-terminus antibodies. (e) WT and APP knockout (KO) forebrain lysates obtained from 2 month old animals were immunoprecipitated with Y188, H-90, or rabbit IgG and immunoblotted with 22C11 and N-17 for endogenous APP and Plk2 detection in vivo. (f) Coomassie blue staining of bacterially purified proteins as indicated. (g) Purified GST-Plk2c and MBP-APPc were incubated, pulled down with glutathione sepharose, and immunoblotted using GST and APP C-terminus antibodies. Inputs, 5% of lysates. (h) Surface plasmon resonance assay was performed with purified GST-Plk2c and MBP-APPc, or negative control MBP. Binding response (relative units, RU) showed robust interaction between immobilized GST-Plk2 and injected MBP-APPc but not MBP alone. Background was defined as MBP-APPc binding to GST alone, which was subtracted from the values shown. Bar indicates protein injection. Molecular weights in kDa. Experiments were performed in at least duplicate. Cropped blots are displayed for clear and concise presentation. Full blots are shown in Supplementary Fig. 10.