Fig. 3. The effect of CRB3 overexpression on the biogenesis of tight junctions and adherens junctions. (A) Parental MDCK cells were initially cultured in low calcium media. At t=0, the low calcium media was replaced with normal calcium media. At the indicated timepoints, cells were fixed, permeabilized, and co-immunostained with anti-ZO-1 and anti-E-cadherin. (B) The same calcium switch experiment was performed on MDCK cells expressing the indicated Myc-CRB3 proteins. At the indicated times after re-addition of calcium, cells were stained with anti-ZO-1 (green) and anti-E-cadherin (red). ZO-1 and E-cadherin are markers of the TJ and adherens junction, respectively. (C) MDCK cells expressing either Myc-CRB3 or Myc-CRB3ERLI were immunostained with anti-Pals1 (green) and anti-ZO-1 (red) antibodies at 6 hours post-calcium switch.