Supplemental Material

This article contains the following supporting material:

• Supplemental Figure S1 - Specificity of hDlg1, MPP7 and CASK antibodies. (A) Lysates from MCF7 cells treated with control, hDlg1-, CASK- or MPP7-siRNA duplexes for 72 hours were western blotted for hDlg1, MPP7 and CASK and α-Tubulin as a loading control. Molecular weight (MW) markers are indicated on the right. Myc-tagged MPP3 and MPP7 constructs were transfected into HEK 293T cells. Equal amounts of total cell lysates were immunoblotted with MPP7, myc and α-Tubulin antibodies, as indicated.
  (B) MCF7 cells were fixed and stained with affinity-purified rabbit polyclonal MPP7 antibodies. As a control, anti-MPP7 was competed with the MPP7 peptide used for immunization or the corresponding pre-immune IgGs were used. (C) MCF7 cells treated with control or MPP7 siRNA duplexes for 72 hours were fixed and stained with antibodies to MPP7. Note the untransfected cells still showing MPP7 localization at the plasma membrane (arrowhead). All experiments shown in Supplementary Figure S1 are representatives of three independent experiments.
• Supplemental Figure S2 - Details on the MS/MS identification of CASK and MPP7. Indicated in this table are the peptides (and their positions within the protein) that were unambiguously identified following MASCOT database searching. The score MASCOT gave to the corresponding MS/MS spectrum and its threshold value (set at the 95% confidence level) is indicated in the last two columns.
• Supplemental Figure S3 - Plasma membrane localization of MPP7 deletion mutants. Myc-tagged MPP7 deletion constructs were transiently expressed in MCF7 cells for 30 hours. After fixation, cells were stained with anti-myc antibodies and anti-Occludin antibodies to visualize cell junctions. The appropriate constructs are shown above each panel. All experiments shown in Supplementary Figure S3 are representatives of three independent experiments.
• Supplemental Figure S4 - hDlg1 and MPP7 are necessary for tight junction integrity. (A) Transepithelial electrical resistance (TER) measurements of Caco-2 cells stably expressing Ctrl-, hDlg1-A- and hDlg1-B-shRNA as indicated. Caco-2 cell lines were seeded onto Transwell filters and grown at confluence for 5 days. After incubation in low-calcium medium for 16 hours, the cells were incubated in normal growth medium, and the restoration of cell junctions was monitored by measuring TER, expressed in Ohms/cm². Shown are the absolute values of one experiment representative of three independent experiments performed in triplicate. (B) Transepithelial electrical resistance (TER) measurements of Caco-2 cells stably expressing Ctrl-, MPP7-A- and MPP7-B-shRNA as indicated. Measurements and cell culture was performed as described in (A).