Journal: Molecular Biology of the Cell

Article Title: Tight Junction–associated MARVEL Proteins MarvelD3, Tricellulin, and Occludin Have Distinct but Overlapping Functions

doi: 10.1091/mbc.E09-08-0734

Figure Lengend Snippet: TAMPs demonstrate unique interactions with each other and ZO-1. (A) Coimmunoprecipitation of TAMPs from Caco-2 lysates reveals interactions among marvelD3, tricellulin, and occludin. The data are consistent with a model of TAMP interaction, where marvelD3 physically associates with both occludin and tricellulin, but the latter two TAMPs do not interact. (B) TAMP cytoplasmic tails were expressed as GST fusion constructs, and equal quantities of each were loaded onto GST-agarose, as confirmed by the Coomassie blue–stained gel. Pulldown experiments using VSVG-ZO-1-GuK as prey demonstrate interaction with the carboxy-terminal tail of occludin (immunoblot). In contrast, neither cytoplasmic tails of tricellulin nor marvelD3 were able to pull down VSVG-ZO-1-GuK. Data are representative of ≥2 experiments, each in duplicate, with similar results.

Article Snippet: Glutathione S -transferase (GST) and VSVG (vesicular stomatitis virus glycoprotein) fusion constructs were generated by joining cDNA sequences into pGEX-4T3 (GE Healthcare Bio-sciences, Piscataway, NJ), and removing the GST coding sequence by site-directed mutagenesis (Stratagene, La Jolla, CA) for VSVG constructs.

Techniques: Construct, Staining, Western Blot

Journal: Scientific Reports

Article Title: IC-Tagging methodology applied to the expression of viral glycoproteins and the difficult-to-express membrane-bound IGRP autoantigen

doi: 10.1038/s41598-018-34488-3

Figure Lengend Snippet: Characterization of protein re-localization to sec-muNS-Mi* MS inside the ER promoted by IC*-tagging. ( a ) Immunofluorescence analysis of DF-1 cells transfected with sec-muNS-Mi* (sec-Mi* in the figure) and co-transfected either with sec-mRFP (upper row) or sec-mRFP-IC* (middle and lower row). mRFP fluorescence is seen in red, while sec-muNS-Mi* is shown in green after detection with muNS-specific antibodies. Nuclei were stained blue with DAPI and shown in the merged images. The white bar represents a distance of 5 µm. The pictures of the upper and middle row were taken with an epi-fluorescence microscope (epi on the left of the figure). The lower row shows pictures of a slice acquired with a confocal microscope. ( b ) Confocal microscopy analysis of DF-1 cells transfected with sec-muNS-Mi* and co-transfected with a chimera formed by the ectodomain of the glycoprotein of VSV fused to mRFP and the IC-Tag (VSV-G). The presence of sec-muNS-Mi* was revealed by using muNS-specific antibodies (green). The VSV chimera was detected by the mRFP fluorescence. Nuclei were stained blue with DAPI and shown in the merged image. The white bar represents a distance of 5 µm. ( c ) Western-blot analysis performed with a mix of antibodies against the SV5 epitope and muNS. The samples came from extracts from DF-1 cells transfected with expression plasmids for a VSV chimera similar to the one described in b, but where the mRFP was replaced by the SV5 epitope (1 and 2) or co-transfected with sec-muNS-Mi* (3 and 4) either before (1 and 3) or after (2 and 4) treatment with N-glycosidase.

Article Snippet: The ectodomain of the VSV glycoprotein (VSV-G) was obtained from plasmid pCMV-VSV-G (gift from Bob Weinberg, Addgene plasmid # 8454) by PCR amplification and cloned into the plasmid pcDNA 3.1 Zeo+ to generate pcDNA sec-VSVG.

Techniques: Immunofluorescence, Transfection, Fluorescence, Staining, Microscopy, Confocal Microscopy, Western Blot, Expressing