Journal: PLoS ONE
Article Title: A Membrane Fusion Protein ?SNAP Is a Novel Regulator of Epithelial Apical Junctions
Figure Lengend Snippet: GBF1, but not other Golgi GEFs, regulates barrier functions of the intestinal epithelial junctions. ( A ) Immunoblotting analysis demonstrates that two different αSNAP-specific siRNA duplexes significantly decrease expression of Golgi GEFs, GBF1, BIG1 and BIG2 in SK-CO15 cells on days 2 and 3 post-transfection. ( B,C ) SK-CO15 cells were transfected with either control GBF1, BIG1, or BIG2, siRNAs and examined on day 4 post-transfection. Immunoblotting analysis demonstrates selective down regulation of individual GEFs by their gene-specific siRNAs. Depletion of GBF1, but not BIG1 or BIG2, decreased p120 catenin expression ( B ), dramatically inhibited development of the paracellular barrier ( C ) and induced apoptosis ( B ); *p
Article Snippet: Antibodies and other reagents The following primary polyclonal (pAb) and monoclonal (mAb) antibodies were used to detect junctional, signaling and vesicle trafficking proteins: anti-αSNAP mAb (Abcam) anti-occludin, ZO-1, claudin-1, claudin-4, and JAM-A mAbs and pAbs (Invitrogen, Carlsbad, CA); anti-cleaved PARP, active caspase-3, active caspase-7 and GAPDH pAbs (Cell Signaling); anti-E-cadherin, β-catenin, GM-130 and NSF mAbs (BD Biosciences, San Jose, CA); anti-ERGIC53 mAb (Enzo Life Sciences, Plymouth Meetings, PA); anti-BIG1 and Giantin pAbs (Covance, Princeton, NJ); anti-β-catenin pAb (Sigma-Aldrich, St. Louis, MO); anti-α-catenin mAb (Epitomics, Burlingame, CA).
Techniques: Expressing, Transfection