Full Length Jam Expression Vector, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM)"
Article Title: The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM)
Journal: The EMBO Journal
Figure Legend Snippet: Fig. 3. JAM associates with ASIP generated in COS-7 cells. COS-7 cells were transiently transfected with either an irrelevant expression vector encoding bacterial alkaline phosphatase (mock), N-terminal T7 tag-fused ASIP expression constructs that encode full-length ASIP (ASIP fl) or deletion mutants comprising amino acid residues 583–1337 (ASIP 583) or 258–936 (ASIP 258). Lysates from transfected cells were incubated with GST–JAM, GST–JAMΔ3 or GST alone, immobilized on G–Sepharose beads. The resulting complexes were analysed by immunoblotting with a mAb directed against the T7 tag. Arrowheads indicate the positions of the recombinant ASIP molecules. Both full-length ASIP and ASIP deletion mutant ASIP 258 were efficiently affinity isolated with GST–JAM, whereas the ASIP mutant lacking the first PDZ domain (ASIP 583) was only poorly affinity isolated.
Techniques Used: Generated, Transfection, Expressing, Plasmid Preparation, Construct, Incubation, Recombinant, Mutagenesis, Isolation
Figure Legend Snippet: Fig. 6. JAM recruits endogenous ASIP to cell–cell contacts in transfected CHO cells. ( A ) Western blot analysis of ASIP and ZO-1 in CHO and CMT cells. Arrowheads indicate the 100 and 180 kDa isoforms for ASIP and the 220 kDa isoform for ZO-1. ( B ) Double-label immunofluorescence analysis of CHO cells transfected with JAM. Heterogeneous CHO cell populations consisting of transfected cells and non-transfected cells were simultaneously stained with rat anti-JAM mAb and rabbit pAb directed against ASIP or ZO-1, or mouse mAb directed against HSP-90. Incubation with primary antibodies was followed by biotinylated secondary antibodies and Cy-3-conjugated streptavidin to detect JAM, and with Cy-2-conjugated secondary antibodies to detect ASIP, ZO-1 and HSP-90. In JAM-transfected CHO cells JAM clustered at sites of cell–cell contact between transfected cells (arrowheads), but not between transfected and non-transfected cells (thin arrows), indicating that homophilic interaction is required for junction localization. ASIP and ZO-1 co-localized with JAM at sites of cell–cell contacts. Bar, 5 µm.
Techniques Used: Transfection, Western Blot, Immunofluorescence, Staining, Incubation
Figure Legend Snippet: Fig. 5. ASIP and JAM associate in mammalian epithelial cells. MDCK II cells transfected with empty vector, expression vector encoding Flag-JAM or Flag-JAMΔ9 were subjected to a Ca 2+ switch for 1 h, then lysed in the presence or absence of a chemical cross-linker. Lysates were incubated with affinity-purified ASIP antibodies, and the resulting immunocomplexes were analysed using the antibodies indicated on the left. To test for the amounts of protein present in each sample, aliquots of each lysate were removed prior to immuno precipitation, and tested for the presence of endogenous ASIP and aPKCλ as well as of transfected JAM (Sup). Co-immunoprecipitations in the absence and presence of the cross-linker are shown in the middle and right panels, respectively. The signals corresponding to the two ASIP isoforms present in MDCK II cells (180 and 150 kDa isoforms) tend to be diffuse in samples subjected to cross-linking, probably because of incomplete cleavage of the cross-linker. The asterisk indicates a high molecular weight band that was specifically detected in immunocomplexes derived from Flag-JAM-expressing cells. Note that Flag-JAM, but not Flag-JAMΔ9, is co-precipitated with ASIP and that this requires the presence of a cross-linker, whereas aPKCλ is co-precipitated with ASIP in all samples.
Techniques Used: Transfection, Plasmid Preparation, Expressing, Incubation, Affinity Purification, Immunoprecipitation, Molecular Weight, Derivative Assay
Figure Legend Snippet: Fig. 7. Overexpression of the cytoplasmic domain of JAM results in the loss of the junctional staining of ASIP in MDCK II cells. Subconfluent MDCK II cells were transiently transfected with expression vectors encoding Flag-tagged mutants of JAM encoding the transmembrane and cytoplasmic domain of JAM, with the complete C-terminus (JAM M/C) or a truncated C-terminus lacking the last nine amino acid residues (JAM M/CΔ9). Cells were immunostained with anti-Flag antibody M2 (A–C) together with anti-ASIP (B), or anti-ZO-1, anti-aPKCλ and anti-β-catenin antibodies (C). ( A ) Confocal z -sectional views of cells expressing the JAM mutants. Note that both mutants show a similar distribution along the whole cellular surface. ( B ) JAM-transfected cells appear as bright cells when stained with the anti-FLAG mAb (left panels). Cells overexpressing JAM M/C but not JAM M/CΔ9 lack the junctional staining of ASIP at cell–cell borders (right panels). Based on ASIP localization at cell junctions, cells were classified as complete (complete loss of ASIP, black bars), partial (partial loss, middle grey bars) or intact (intact ASIP staining, dark grey bars). Cells with aberrant shape are represented by light grey bars. Statistical data represent mean values of three independent experiments with a minimum of 200 cells counted for each sample. ( C ) Cells overexpressing JAM M/C also lack aPKCλ as well as ZO-1 but not β-catenin staining at cell–cell junctions. Note that ZO-1 often forms aggregates in cells expressing JAM M/C but not JAM M/CΔ9. Bars, 15 µm.
Techniques Used: Over Expression, Staining, Transfection, Expressing
Article Snippet: For stable transfections, 1 × 107 L cells were transfected with 20 µg of full-length JAM expression vector (pFLAG- CMV-1) and 3 µg of hygromycin-resistance vector (pPGKHyg) by electroporation (0.25 kV, 950 µF), and then cultured in the presence of 900 µg/ml