G12 regulates protein interactions within the MDCK cell tight junction and inhibits tight-junction assembly

JCS014878 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Gα12 or QLα12 expression does not alter tight junction protein levels. Lysates were prepared from WTα12- and QLα12-MDCK cells cultured in +/−dox for 72 hours as described in Materials and Methods. Lysates (50 µg) were analyzed by SDS-PAGE and western blot for Gα12 and TJ proteins as described. For ZO-1, ZO-2 and occludin the blot was stripped and reprobed. For the remaining western blots, separate gels were analyzed.

• Supplemental Figure S2 -

  Fig. S2. Activated Gα12 regulates MDCK cell barrier function through a Src-dependent mechanism. (A) Transepithelial Resistance was measured as described in Materials and Methods for Gα12 and QLα12-MDCK cells +/−dox for 72 hours and the Src inhibitor PP2 (10 µM) added at the time of changing to −dox medium. (B) Sodium and chloride permeability in QLα12-MDCK cells +/−dox and +PP2 were measured in Ussing chambers as described in Materials and Methods. (C) Size selectivity for cation permeability in QLα12-MDCK cells +/−dox and −dox+PP2 (10 µM) was determined relative to sodium. Lithium (Li; 1.2 A), potassium (K; 2.66 A), rubidium (Rb; 2.96 A), cesium (Cs; 3.38 A) and arginine hydrocholoride (6.96 A).

• Supplemental Figure S3 -

  Fig. S3. Activated Gα12 promotes redistribution of claudin-1 to intracellular sites. (A) Immunofluorescent microscopy of QLα12-MDCK cells +/−dox and −dox +PP2 co-stained for ZO-1 (Texas red) and claudin-1 (FITC) with merged images in last column. (B) Western blot for ZO-1 and claudin-1 from QLα12-MDCK cells cultured in +dox, −dox, −dox +PP2 and −dox +GA separated in to soluble (S) and insoluble (I) detergent fractions as described in Materials and Methods.