(A) shows total β-Arrestin1 and ARHGAP21 expression in Caco-2 ShPTEN cell lysates (top two panels) after PTEN C2 (C2), C2-MCBR3 or EV control transfections. Expression levels of transfected GFP-labelled proteins and β-Actin loading controls shown in lower two panels. (B) indicates summary fold changes of total β-Arrestin1; C2 = 1.23 ± 0.02; **p<0.01; MCBR3 = 1.07 ± 0.02; [MCBR3 vs control = NS]. (C) shows respective fold changes of ARHGAP21; C2 = 0.8 ± 0.02; **p<0.01; MCBR3 = 0.95 ± 0.03; [MCBR3 vs control = NS; all values in ADU]. (D) Effects of transfections on cytosol and membrane β-Arrestin1 and ARHGAP21 in Caco-2 ShPTEN (ShPTEN) cells (top two panels). Expression levels of transfected GFP-labelled proteins and E-Cadherin and HSP90 membrane and cytosolic markers shown in lower three panels. (E, F) Fold change of β-Arrestin1 and ARHGAP21 expression induced in the membrane fraction. (E) β-Arrestin1, C2 = 1.25 ± 0.05; *p=0.04 and (F) ARHGAP21 = 0.53 ± 0.09; *p=0.03. β-Arrestin1 and ARHGAP21 ADU values normalized against total lysate ADU for each protein. (G) β-Arrestin1-associated ARHGAP21 induced in ShPTEN membrane fractions by C2, C2-MCBR3 vs EV control transfections (top panel). β-Arrestin1 bait signal shown in second panel. Total β-Arrestin1 and ARHGAP21 in lysates shown in third and fourth panels. Expression of GFP-labeled C2, C2-MCBR3, EV and E-Cadherin membrane marker shown in lower three panels. (H) Fold changes of β-Arrestin1-associated ARHGAP21 normalized against total ARHGAP21 in the membrane fraction (ADU) - C2 = 6.27 ± 0.51; ***p<0.001; MCBR3 = 1.25 ± 0.15; [MCBR3 vs control = NS]. (I) β-Arrestin1-associated ARHGAP21 ADU after expression of C2, C2-MCBR3, wt PTEN or PTEN-MCBR3 vs EV control in PTEN -/- cell membrane fractions (top panel). β-Arrestin1 bait signal, total lysate expression of each protein, expression of GFP-labelled EV or C2 domain constructs and E-Cadherin membrane marker shown in lower five panels. (J) Summary fold changes of β-Arrestin1-associated ARHGAP21 normalized against total membrane ARHGAP21; C2 = 7.33 ± 0.64; C2-MCBR3 = 2.5 ± 0.35; PTEN = 12.93 ± 0.19; PTEN MCBR3 = 3.15 ± 0.21 ADU; control vs C2 or PTEN, ***p<0.001; control vs C2-MCBR3 (NS). Control vs PTEN-MCBR3 = *p<0.05. Analyses by ANOVA, Tukey post hoc or Student’s paired test. Molecular weights indicated by arrows in blots.