Structure-function analysis of Par3 requirement for E4orf4’s toxicity in HEK 293T cells. (A) Flag-E4orf4 IP prepared from 293T cells transfected with the vector (EV) or Flag-E4orf4-mRFP, followed by IB for Par3 isoforms (arrows), and Par6; the levels of Flag-E4orf4 and Par6 in total cell extracts (Input), and GAPDH as a loading control, are shown. (B) GFP and GFP-Par3 IPs prepared from 293T cells transfected with E4orf4-mRFP together with GFP or GFP-Par3, followed by IB for the indicated antibodies; E4orf4 levels in total cell extracts are shown (Input). (C) Schematic representation of Par3-GFP proteins with functional domain deletions. (D) Representative IB showing the efficiency of Par3 depletion in 293T-shPar3 cell lines, estimated by loading increasing amounts of control cell extracts; vinculin is shown as a loading control. (E and F) Flag-E4orf4 IPs, or GFP-Par3 IPs, prepared from 293T-shPar3A#4 cells transfected with Flag-E4orf4-mRFP and GFP-Par3 constructs, followed by IB with the indicated antibodies; FL, full length GFP-Par3; n > 2; levels of E4orf4-mRFP and GFP-Par3 in total cell extracts are shown (Input). (G) IB showing the levels of GFP-Par3 proteins from a representative depletion-rescue experiment; GAPDH is shown as loading control. (H) Confocal images of 293T-shCTL and 293T-shPar3A#4 cells transfected with E4orf4-mRFP showing loss of E4orf4-dependent cell blebbing in Par3-depleted cells; F-actin is stained with phalloidin. Bar: 10 µm. (I) Quantification of the percentage of cell blebbing and nuclear condensation cells, as scored relative to cells expressing E4orf4-mRFP 24 h post-transfection for the datasets represented in H; data are from one representative experiment, or means ± SEM [n = 3]). (J) Functional analysis of the molecular requirements of Par3 for E4orf4-induced cell blebbing and nuclear condensation by depletion-rescue (means ± SEM [n ≥ 737 cells; n ≥ 4]). ***, P < 0.001; ****, P < 0.0001; ns, not significant.