Figure S3.

Requirement of the binding of afadin to αE-catenin for the proper localization of AJ proteins. (A–D) Localization of myosin IIB, phosphorylated myosin light chain II, and FAB proteins in afadin-KO cells expressing FLAG-tagged afadin, FLAG-tagged afadin-ΔCC, or FLAG-tagged CC-afadin. Afadin-KO cells were expressed with FLAG-tagged afadin, FLAG-tagged afadin-ΔCC, or FLAG-tagged CC-afadin. The cells were fixed and stained with the indicated Abs and phalloidin, followed by immunofluorescence microscopic analysis. (A) E-cadherin and myosin IIB; (B) E-cadherin and phosphorylated myosin light chain II (phospho-MLC); (C) E-cadherin and α-actinin; and (D) E-cadherin and vinculin. Arrows indicate the F-actin signal at bicellular AJs. The results are representative of three independent experiments. (E) Quantification by line-scans of the immunofluorescence images of FLAG-afadin, FLAG-afadin-ΔCC, and FLAG-CC-afadin cells in A–D. Statistical analysis was performed as described in the legend to Fig. 1. The ratios of FWHM of proteins of interest to FWHM of E-cadherin are indicated. Data are expressed as a box-and-whisker plot of three independent experiments (n = 10). The whiskers indicate the maximum and minimum values, and the box corresponds to the 75th percentile, median, and 25th percentile values. *, P < 0.01 (two-tailed, unpaired Student’s t test). N.S., not significant.