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Molecular Biology of the Cell: E03-07-0531

This article contains the following additional material:

Supplemental Figure PDF

  • Supplemental Figure Detergent extraction of KLEIP, alpha-catenin, beta-catenin and E-cadherin from MDCK cells after the calcium switch. MDCK cells grown on 10-cm dished were subjected to the calcium switch assay (0 hr, 8 hr and 24 hr) as described in the material and method. Cells were washed with PBS containing 1mM CaCl2 and MgCl2, collected by scraping, and then centrifuged at 2,000 x g for 3 min. Cells were lysed for 10 min on ice in 320 mul 0.5% Triton-X100 extraction buffer (PBS containing 0.5% Triton-X100 (v/v), 1mM CaCl2 and 1mM MgCl2). The lysates were centrifuged at 14,000 x g for 15 min to obtain the soluble fraction. To the supernatant, 80 mul of 5 x Laemmli buffer was added. The pellets (the insoluble fraction) were resuspended in 400 mu1 laemmli buffer. Equivalent aliquots of supernatants and pellets were analyzed by immunoblotting by anti-KLEIP, anti-alpha-catenin, anti-beta-catenin and anti-E-cadherin. S and I represent soluble and insoluble fractions, respectively.

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