Anti Mhcii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The intramembrane protease SPPL2a promotes B cell development and controls endosomal traffic by cleavage of the invariant chain"
Article Title: The intramembrane protease SPPL2a promotes B cell development and controls endosomal traffic by cleavage of the invariant chain
Journal: The Journal of Experimental Medicine
Figure Legend Snippet: Disturbance of membrane traffic within the endocytic system of SPPL2a −/− B cells. (A and B) Visualization of CD74 in isolated splenic SPPL2a +/+ and SPPL2a −/− B cells by indirect immunofluorescence using an antibody against an N-terminal epitope detecting the NTF and the full-length protein. EEA1 (A) and LAMP1 (B) served as markers of early endosomes and lysosomes/late endosomes, respectively. Bars, 2 µm. (C) Transmission electron microscopy of splenic IgM + B cells from wild-type or SPPL2a −/− mice. Bars, 1 µm. (D) Vacuoles in SPPL2a −/− B cells exhibited various contents of low-electron density. Occasionally, multivesicular bodies (mvb) were observed (arrow). Bar, 500 nm. (E) Presence of CD74 NTF, LAMP1, and MHCII in vacuoles of IgM + B cells from SPPL2a −/− mice was assessed by immunogold labeling. Bars, 200 nm (CD74 and MHCII single labeling) or 100 nm (CD74 + LAMP1 double labeling). (F and G) Surface and total MHCII levels in transitional stage T1 B cells (B220 + CD21 low CD24 high ) of SPPL2a-deficient or wild-type mice. Splenocytes were stained for B220, CD21, and CD24, allowing for identification of B cell subsets. Subsequently, cells were incubated with anti-MHCII with or without previous permeabilization and analyzed by flow cytometry. Surface and total MHCII levels are shown as histograms representative of three independent experiments or as mean of median fluorescence intensity (MFI) from three mice per genotype (G). ***, P
Techniques Used: Isolation, Immunofluorescence, Transmission Assay, Electron Microscopy, Mouse Assay, Labeling, Staining, Incubation, Flow Cytometry, Fluorescence
Figure Legend Snippet: The intramembrane protease SPPL2a cleaves CD74 NTF. (A) Scheme of proteolytic degradation of CD74 in MHCII compartments, where the luminal domain is removed in a stepwise fashion by endosomal proteases and finally released from the MHCII dimer by cathepsin S. A small fragment (CLIP) persists inside the peptide-binding groove of MHCII, which is subsequently replaced with an antigenic peptide before the MHCII–peptide complex is transported to the plasma membrane. The remaining transmembrane NTF (82 aa) of CD74 is then proteolyzed by SPPL2a. The catalytically critical YD and GxGD motifs of SPPL2a are indicated by colored asterisks. (B) HEK293 cells stably expressing the p31 isoform of CD74 (HA-CD74p31-V5) were treated with 10 µM (Z-LL) 2 -ketone, 1 µM inhibitor X, 100 µM leupeptin or 25 mM NH 4 Cl for 5 h. The CD74 NTF is indicated by the open arrowheads. Full-length CD74 (closed arrowheads) and CD74 NTF were detected with anti-HA recognizing the epitope tag fused to the N terminus of the protein. (C) Transient knockdown of SPPL2a in HEK293 cells stably expressing HA-tagged CD74 (HA-CD74p31-V5). SPPL2a and the lysosomal membrane protein LAMP2 as control were analyzed in carbonate-washed membranes from the same batch of cells for enhancing SPPL2a detectability. (D) SPPL2a or the inactive D416A mutant were transiently co-expressed with CD74, followed by detection with anti-CD74 (In-1). (E) Using an antibody against an N-terminal epitope of CD74, endogenous CD74 was analyzed in splenic IgM + B cells isolated from SPPL2a −/− and control mice. (B–E) Electrophoretic separation before detection of CD74 was performed by standard Tris-Glycine SDS-PAGE (D) or using a Tris-Tricine buffer system (B, C, and E) with improved resolution in the low-molecular weight range. Equal protein loading was confirmed as indicated. Data are representative of three independent experiments.
Techniques Used: Cross-linking Immunoprecipitation, Binding Assay, Stable Transfection, Expressing, Mutagenesis, Isolation, Mouse Assay, SDS Page, Molecular Weight
2) Product Images from "A Novel Cellular Pathway of Antigen Presentation and CD4 T Cell Activation in vivo"
Article Title: A Novel Cellular Pathway of Antigen Presentation and CD4 T Cell Activation in vivo
Journal: Frontiers in Immunology
Figure Legend Snippet: Recruited monocytes present antigen and may be observed interacting with T cells in the lymph node at 24 h post-immunization. (A) Representative plots showing flow cytometry gating strategy for the identification of CD11b+CD64+ monocytes, for CD11b+CD169+ SCS macrophages and CD11b- or CD11b+ CD11c+MHCII+ dendritic cells. Graphs showing mean (±SEM) YAe MFI on CD11b- (B) and CD11b+ (C) dendritic cells at 4, 24, and 48 h; mean (±SEM) YAe MFI SCS macrophages (D) at 4 h; mean (±SEM) YAe MFI on monocytes (E) at 4, 24, and 48 h. Groups contained 3 animals and data is representative of three independent experiments. (F) LysM-EGFP or CD11cYFP mice were immunized in the footpad (OVA or BSA/ISCOMATRIX or OVA/LPS respectively) ~20 h later the draining popliteal lymph node was surgically exposed. OTII DSRed T cells and monocytes (GFP+) or DCs (YFP+) were imaged by live real time multiphoton microscopy. Interaction between T cells and monocytes (and T cells and DCs was identified by colocalization of the DSRed signal with the GFP or YFP signals and tracked for up to 20 min, 48 such interactions were tracked in the OVA/ISCOMATRIX treated mice. IMX = ISCOMATRIX™ adjuvant, * P
Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, Microscopy