Fig. 4

GRK6 palmitoylation and kinase activity recruit the membrane localization of β-Arrestin to trigger the EMT progression and cellular migration activity in TNBC cells. A–B. Western blot analyses for the phosphorylated β-Arrestin (p-β-Arrestin), total β-Arrestin, membrane (Mem.)-associated/cytosolic (Cyt.) β-Arrestin 1/2, Na+/K+ATPase and GAPDH in the parental (PT) HCC1937 cells without or with overexpression of vector control (VC), GRK6A or GRK6B (A) and in MDA-MB231 treated with GRK6 inhibitor at the indicated concentrations for 24 h (B). C. Immunoprecipitation assay for GRK6:β-Arrestin complex in the NS/GRK6-sh2 MDA-MD231 cells and VC/GRK6-OE HCC1937 cells. D–F. Western blot analyses for β-Arrestin 1/2 (D) and the EMT markers (E) in the whole cell lysate and Giemsa stain for the migrated cells in the trans-well cultivation (F, upper) of vector control HCC1937 cells and GRK6A-overexpressing HCC1937 cells treated without or with β-Arrestin inhibitor at the indicated concentrations for 24 h. The migration cell number from three independent experiments were presented as mean ± SEM in the histogram (F, lower). GAPDH in A, B, D and E was used as an internal control of total and cytosolic protein loading. Na+/K+ATPase in A, B and C was used as an internal control of membrane protein loading. In E, the different alphabets indicate the statistical significance at p < 0.05