Figure 4.

R200/R201 mediate Caprin-2’s localization to the membrane.A, membrane lipid strips probed with purified 6×His tagged hCap2_HR1 WT, K196E/K197E, or R200E/R201E, respectively. Relative protein levels were quantified using ImageJ software (34). B and C, measurement of the binding affinity of PI4P with hCap2_HR1 WT (B) or with the R200E/R201E double mutant (C). Biotinylated PI4P were loaded onto streptavidin sensors and were then incubated with different concentrations of purified hCap2_HR1 WT (B) or R200E/R201E (C). Curves are shown in colors that are corresponding to sample concentrations as shown in the top right-hand corner. K_dis (dissociation rate) and K_on (on-rate) as well as the corresponding errors were calculated with Octet Data Analysis 11.0. D, subcellular localization analysis of Caprin-2 WT and R200E/R201E mutant. Endogenous β-catenin was used as an indicator of the plasma membrane. Flag-tagged full-length Caprin-2 WT or R200E/R201E mutant was transfected into U2OS cells grown on glass coverslips. After being fixed with 4% (w/v) paraformaldehyde and permeabilized with 0.1% (v/v) Triton X-100, anti-Flag, or anti-β-catenin was added followed by secondary antibody treatments. Photos were taken with Leica TCS SP8. Panels (left to right): Caprin2 (green); β-catenin (red); 4′, 6-diamidino-2-phenylindole (blue); merged images. Scale bars represent1 0 μm. HR, homologous region; PI4P, phosphatidylinositol 4-phosphate.