E4orf4 interacts with Par3 and modulates cell polarity signaling. (A) Flag-AP-MS identification of Flag-E4orf4’s partner proteins, as analyzed by the SAINT algorithm. High-confidence interactors were ranked according to their score (graph), and the average spectral count in the control (green) or E4orf4 sample (gray) is shown; three independent experiments are quantified on the y axis. (B) Flag-E4orf4 IP prepared from MDA-MB-231 cells expressing Flag-E4orf4 followed by IB for Par3 isoforms (arrows); the levels of Flag-E4orf4 in total cell extracts (Input) and GAPDH as loading control are shown (representative of n = 3). (C and D) Flag-E4orf4 IPs from 293T-shPar3#4 cells transfected with GFP or GFP-Par3 and Flag-E4orf4-mRFP or from MCF10A cells transduced with lacZ or E4orf4-mCherry (D), followed by IB with the indicated antibodies; Input: total cell extracts; arrows: Par3 isoforms; n = 2. (E) Schematic of the protocol used. (F and H) Merged plane views from confocal image stacks of MCF10A cells transduced with lacZ or E4orf4 showing staining of E-cadherin or ZO-1; F-actin was labeled with phalloidin; N.A., not applicable. The dotted lines in H indicate the position of the y axis presented as y-z views on the right panels; magenta arrowheads indicate ZO-1 clusters induced by E4orf4 (n ≥ 3). Bars: 20 µm. (G) Quantification of the cortical/cytoplasmic ratio of E-cadherin for datasets represented in F; means ± 95% CI; ****, P < 0.0001.