Figure 3. EpCAM restrains claudin-7 on... | Rockefeller University Press

Figure 3.

$EpCAM restrains claudin-7 on the basolateral membranes. (A) Pull-down assay of EpCAM-FLAG. Immunoprecipitates (IPs) from EpCAM-KO MDCK II cells expressing EpCAM-FLAG were compared with those from EpCAM-KO cells by silver staining (left) and immunoblotting using HRP-linked anti-FLAG (M2), mouse anti-EpCAM mAb, and other antibodies (right). Arrowhead and arrow in the left panel indicate EpCAM-FLAG and claudin(s), respectively. Asterisks in the right panels indicate degradation products of claudins. (B) Immunoprecipitation of EpCAM-FLAG from HEK293T cells expressing EpCAM-FLAG and claudin-GFP using 1% NP-40 (upper panels) or 1% Brij97 (lower panels). (C) Immunoprecipitation of EpCAM-FLAG from HEK293T cells expressing EpCAM-FLAG and claudin-GFP with or without claudin-7-mCherry using 1% Brij97. Asterisk indicates the heavy chain of IgG. (D) Fractionation of EpCAM-FLAG–expressing EpCAM-KO MDCK II cells treated with various concentrations of trypsin into Brij97-soluble and insoluble fractions and immunoprecipitation of EpCAM-FLAG from the soluble fraction. (E) Predicted structure of the EpCAM–claudin-7 complex. Side views of the complex from three directions are shown. Red and orange indicate the regions before and after the putative cleavage site of EpCAM, respectively. Light blue, salmon, yellow, and pale green indicate the first extracellular loop, second extracellular loop, transmembrane region, and intracellular region of claudin-7, respectively. Out, extracellular space. In, cytoplasmic space. Mem, plasma membrane. (F) Predicted structure of cleaved EpCAM and claudin-7. Black arrow indicates the exposed first extracellular loop of claudin-7. (G) The model of paired claudin-15 molecules (white and teal), superimposed with full-length (red and orange) and cleaved (orange) EpCAM. Black arrow indicates that orange EpCAM molecule and teal claudin molecule cause steric hindrance. Source data are available for this figure: SourceData F3.$

EpCAM restrains claudin-7 on the basolateral membranes. (A) Pull-down assay of EpCAM-FLAG. Immunoprecipitates (IPs) from EpCAM-KO MDCK II cells expressing EpCAM-FLAG were compared with those from EpCAM-KO cells by silver staining (left) and immunoblotting using HRP-linked anti-FLAG (M2), mouse anti-EpCAM mAb, and other antibodies (right). Arrowhead and arrow in the left panel indicate EpCAM-FLAG and claudin(s), respectively. Asterisks in the right panels indicate degradation products of claudins. (B) Immunoprecipitation of EpCAM-FLAG from HEK293T cells expressing EpCAM-FLAG and claudin-GFP using 1% NP-40 (upper panels) or 1% Brij97 (lower panels). (C) Immunoprecipitation of EpCAM-FLAG from HEK293T cells expressing EpCAM-FLAG and claudin-GFP with or without claudin-7-mCherry using 1% Brij97. Asterisk indicates the heavy chain of IgG. (D) Fractionation of EpCAM-FLAG–expressing EpCAM-KO MDCK II cells treated with various concentrations of trypsin into Brij97-soluble and insoluble fractions and immunoprecipitation of EpCAM-FLAG from the soluble fraction. (E) Predicted structure of the EpCAM–claudin-7 complex. Side views of the complex from three directions are shown. Red and orange indicate the regions before and after the putative cleavage site of EpCAM, respectively. Light blue, salmon, yellow, and pale green indicate the first extracellular loop, second extracellular loop, transmembrane region, and intracellular region of claudin-7, respectively. Out, extracellular space. In, cytoplasmic space. Mem, plasma membrane. (F) Predicted structure of cleaved EpCAM and claudin-7. Black arrow indicates the exposed first extracellular loop of claudin-7. (G) The model of paired claudin-15 molecules (white and teal), superimposed with full-length (red and orange) and cleaved (orange) EpCAM. Black arrow indicates that orange EpCAM molecule and teal claudin molecule cause steric hindrance. Source data are available for this figure: SourceData F3.