Fig. 1. Overexpression of human Crumbs homologue 3 in MDCK renal epithelial cells. (A) Lysates were prepared from the parental MDCK cells, MDCK stable cell line that overexpresses Crumbs 3 (CRB3) or Myc-CRB3, and cells moderately expressing Myc-CRB3. Lysates proteins (left panels) and anti-CRB3 immunoprecipitates (right panel) were resolved via SDS-PAGE and the indicated immunoblots were performed. (B-E) The indicated cell lines were initially cultured on Transwell filters in low calcium media (5 µM Ca²⁺). Subsequently, the media was replaced with normal calcium media (1.8 mM Ca²⁺). Cells were grown in this media for 12 hours and immunostained as indicated according to Materials and Methods. Ezrin and gp135 are markers of the apical surface, whereas E-cadherin marks the lateral surface. Square and rectangular panels represent X-Y photomicrographs and X-Z series (Z-sections), respectively.