CD2AP is required for actin stability at the adherens junction of polarized MDCK epithelial cells. (a) Projection of 40 deconvolved optical z slices spanning the apical 8 µm of cells showing decreased actin accumulation (yellow arrowheads) at cell–cell contacts in CD2AP knockdown cells (white asterisks) stained for E-cadherin, CD2AP, actin (phalloidin), and DNA (DAPI). (b) Western blots of total cell extracts showing normal levels of E-cadherin, α-actinin-4, and actin in stable CD2AP knockdown cell lines, ShCD2AP1 and ShCD2AP2. Molecular weight markers are 150 kD for CD2AP, α-actinin-4, and E-cadherin blots and 50 kD for the actin blot. (c) Single deconvolved optical z slice at the apical region of cells showing knockdown of CD2AP (white arrowheads and outlines) within a monolayer of cells expressing normal levels of CD2AP (yellow arrowheads and outlines). (d) Quantitation of junctional staining of E-cadherin and CD2AP showing a correlation between CD2AP levels and E-cadherin levels in parental but not CD2AP knockdown cells. R is the correlation coefficient. Representative data from four separate experiments are shown. (e) Quantitation of junctional staining of CD2AP and actin (phalloidin) showing a correlation between CD2AP levels and actin levels in both parental and CD2AP knockdown MDCK cells. Representative data from four separate experiments (n = 4) are shown. (f) Quantitation of junctional staining of E-cadherin and actin (phalloidin) showing a lack of correlation between E-cadherin levels and actin levels in both parental and CD2AP knockdown MDCK cells. Representative data from four separate experiments (n = 4) are shown. (g) Projection of 10 deconvolved optical z slices spanning the apical 2 µm of cells showing decreased actin levels at latrunculin-resistant puncta in CD2AP knockdown cells. (h) Quantitation of actin levels at latrunculin-resistant puncta in parental and CD2AP knockdown cells (P < 0.0001). Representative data from four separate experiments (n = 4) are shown.