Identification of two actin-regulating activities at the adherens junctional complex. (a–c) Flow chart represents the overall scheme for biochemical analysis of actin-regulating factors on junction-enriched membranes. (a) Identification of a detergent extractable inhibitory factor on native membranes. Addition of Alexa Fluor 647–labeled monomeric actin to native membranes results in actin assembly associated with the junctional complexes (white arrowheads). Treatment of native membranes with zwitterionic detergent CHAPS or nonionic detergent TX-100 yielded brighter actin puncta (orange and red arrowheads, respectively). (b) Identification of a high-salt/detergent extractable activating factor on native membranes. Treatment of native membranes with high salt followed by CHAPS diminished actin incorporation at membrane puncta (blue arrowheads). Rescaling of the original image shows the presence of very dim fluorescent actin puncta (yellow arrowheads). (c) Identification of a high-salt extractable inhibitory factor on native membranes. Treatment of native membranes with high salt increased actin incorporation at membrane puncta (green arrowheads) when compared with native membranes (purple arrowheads) supplemented with α-actinin-4. (d) Quantitation of actin incorporation at junctional puncta in untreated native membranes and membranes treated with detergents CHAP (P < 0.0001) or TX-100 (P < 0.0001). (e) Quantitation of actin incorporation at junctional puncta in untreated native membranes and membranes treated with high salt (P < 0.0001). (f) Quantitation of actin incorporation at junctional puncta in high salt–treated or sequentially high salt– and CHAPS-treated membranes supplemented with α-actinin-4 (P < 0.0001).