Functional suppression of E-cadherin by protein kinase C

JCS035469 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. GFP-PKCδ expression does not affect the expression level of E-cadherin at the cell surface. MDCK cells stably expressing GFP, GFP-PKCδ or its mutants were subjected to cell surface biotinylation with sulfo-NHS-biotin. For measurement of the cell surface level of E-cadherin, equal amounts of cell lysates were incubated with avidin-immobilized agarose beads and the complexes were detected by immunoblotting with anti-E-cadherin (clone 34). For measurement of the total level of E-cadherin, equal amounts of cell lysates were analyzed by immunoblotting with anti-E-cadherin (clone 34) or anti-β-tubulin as a control. Wt, wild type; KD, kinase-deficient mutant; RD, regulatory domain; ΔC1A, mutant with deletion of the C1A domain (residues 159-208); ΔC1B, mutant with deletion of the C1B domain (residues 232-280); ΔC2, mutant with deletion of the C2 domain (residues 1-123); ΔH, mutant with deletion of the hinge region (residues 280-347); ΔH_280−323, mutant with deletion of residues 280-323; ΔH_324−347, mutant with deletion of residues 324-347.

• Supplemental Figure S2 -

  Fig. S2. The ability of the monoclonal anti-E-cadherin (clone DECMA-1) to detect E-cadherin at cell-cell junctions of MDCK cells relies on the presence of Ca²⁺. Detection of E-cadherin by immunofluorescence staining with monoclonal anti-E-cadherin under three conditions. (i) The cells were grown to confluence (control), (ii) the cells were grown to confluence and then treated with 5 mM EGTA in serum-free medium for 30 minutes (Ca²⁺ depletion), and (iii) after EGTA treatment, the medium was replaced by fresh growth medium for 2 hours (Ca²⁺ repletion). Note that the DECMA-1 antibody recognizes the extracellular domain of E-cadherin and the clone 36 antibody recognizes the intracellular domain of E-cadherin.