mouse anti lamp1  (Developmental Studies Hybridoma Bank)

Journal: Bioactive Materials

Article Title: Countering postoperative immune suppression with a self-assembling dendritic cell nanovaccine

doi: 10.1016/j.bioactmat.2026.05.005

Figure Lengend Snippet: BAITs promote DC activation and antigen presentation via enhanced lysosomal processing. (A) Schematic for MHC Ⅰ/Ⅱ immunostaining. DCs were treated with antigens or BAITs (12 h), then incubated in fresh medium (with TGFβ) for 24 h before staining. (B, C) Quantification of surface MHC Ⅰ (B) and MHC Ⅱ (C) on DCs (n = 8; n.s. is P > 0.05, ∗∗∗ is P < 0.001, ∗∗∗∗ is P < 0.0001 by one-way ANOVA with Bonferroni post-hoc test). (D, E) Representative immunofluorescence images of DCs (blue: nuclei; yellow: LAMP1; green: MHC Ⅰ; magenta: MHC Ⅱ). BAIT-treated DCs show strong surface MHC and minimal intracellular antigen signal, whereas antigen-treated DCs show retained antigen (red) and weak surface MHC signal. (F–H) Flow cytometric analysis of MHC Ⅰ (F), MHC Ⅱ (G), and CD80 (H) expression on murine CD11c + DCs after exposure to antigens or BAITs. (I) Quantification of MHC Ⅰ, MHC Ⅱ, and CD80 expression in DCs by flow cytometry (n = 3; ∗∗∗ is P < 0.001, ∗∗∗∗ is P < 0.0001 by two-way ANOVA with Bonferroni post-hoc test). (J) Heatmap of DC activation-related gene expression after treatment with antigens or BAITs, highlighting upregulation of maturation and antigen presentation genes and downregulation of immunosuppressive genes by BAITs (n = 3). Data are presented as mean ± SD.

Article Snippet: After treatment, the cells were washed twice with PBS and fixed in 4% paraformaldehyde for 20 min. For intracellular protein staining, fixed cells were permeabilized with 0.1% Triton X-100 at room temperature for 10 min. Next, the cells were blocked with 2% BSA and incubated with rat CoraLite Plus 488 anti-mouse LAMP1 antibody (1:200) and rabbit anti-mouse pSAP (1:200) antibody overnight at 4 °C.

Techniques: Activation Assay, Immunopeptidomics, Immunostaining, Incubation, Staining, Immunofluorescence, Expressing, Flow Cytometry, Gene Expression

Journal: bioRxiv

Article Title: Molecular basis of Salla Disease: R39C Mutation Effects on the Lysosomal Transporter Sialin

doi: 10.64898/2026.04.20.719580

Figure Lengend Snippet: Representative images of the effects of mutations on the lysosomal localisation of sialin visualised by epifluorescence microscopy. Wild-type (WT) or different mutated human sialin (R39C, R39K, E194A, E262A, E264A and I266A) tagged with EGFP (green) constructs were transiently expressed in HeLa cells by electroporation. After two days, cells were fixed and analysed under fluorescence microscopy using LAMP1 immunostaining (red) to detect late endosomes and lysosomes. The scale bar represents 10 µm.

Article Snippet: Coverslips were then incubated for at least 1 h with mouse anti-LAMP1 antibodies (H4A3; Developmental Studies Hybridoma Bank) at 0.75 μg/mL in blocking buffer, washed, and incubated with Cy3-conjugated donkey antimouse antibodies (Jackson ImmunoResearch) at 1.4 μg/mL or Alexa Fluor 555 goat antimouse antibodies (Invitrogen) at 2μg/mL in the same buffer.

Techniques: Epifluorescence Microscopy, Construct, Electroporation, Fluorescence, Microscopy, Immunostaining

Journal: bioRxiv

Article Title: Molecular basis of Salla Disease: R39C Mutation Effects on the Lysosomal Transporter Sialin

doi: 10.64898/2026.04.20.719580

Figure Lengend Snippet: Mutagenesis experiments on the sialin R39 site. A and B) Wild-type (WT) or different mutated human sialin tagged with EGFP were transiently expressed in HeLa cells by electroporation. After two days, cells were fixed and analysed under fluorescence microscopy using LAMP1 immunostaining to detect late endosomes and lysosomes. In independent experiments, colocalisation was quantified using scatter plots of the sialin and LAMP1 pixel intensities. The graphs show the Pearson correlation coefficients normalised to that of WT (100%) in three different experiments for the mutants, except for R39C, 6 experiments, with 10 cells per experiment. The statistical analysis was performed by a one-sample t-test comparing the mean of the values for each mutant with a hypothetical mean value of 100: *= p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, ****= p ≤ 0.0001). The red dotted line represents a Pearson coefficient of 0.5, above which we consider that sialin is targeted to lysosomes. It is calculated taking into consideration that the mean Pearson correlation coefficient for WT sialin is 0.73. C and D) Transport activity was measured in a whole-cell assay in which sialin was redirected to the plasma membrane by mutation of the lysosomal sorting motif to facilitate transport measurements (L22G L23G). Human L22G L23G sialin tagged with EGFP with/without mutations was transiently transfected by lipofection in HEK 293T cells. Human sialin (control) and its mutants were assayed for [ 3 H]Neu5Ac uptake at pH 5.0 in 3-6 independent experiments. The graphs show the transport activity relative to that of sialin L22G/L23G (100%) The statistical analysis was performed by a one-sample t-test comparing the mean of the values for each mutant with a hypothetical mean value of 100: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, ****= p ≤ 0.0001). E and F) Close-up views of ICH1 interactions in the completed and minimised models of the LO (E) and CO (F) conformations. Residues in ICH1 (green) are shown to interact with TM1 (orange), TM5 (purple), and other parts (grey) of sialin. Hydrogen bonding (green dashed lines), charge-charge (orange dashed lines), and apolar (purple dashed lines) interactions are shown for residues Y261 to L270 and R39. Hydrogen bonds between backbone atoms involved in the formation of alpha-helices are not shown and only polar hydrogens are displayed to improve visibility. Residues A26-C36 of the N-terminus are not shown to improve visibility.

Article Snippet: Coverslips were then incubated for at least 1 h with mouse anti-LAMP1 antibodies (H4A3; Developmental Studies Hybridoma Bank) at 0.75 μg/mL in blocking buffer, washed, and incubated with Cy3-conjugated donkey antimouse antibodies (Jackson ImmunoResearch) at 1.4 μg/mL or Alexa Fluor 555 goat antimouse antibodies (Invitrogen) at 2μg/mL in the same buffer.

Techniques: Mutagenesis, Electroporation, Fluorescence, Microscopy, Immunostaining, Activity Assay, Clinical Proteomics, Membrane, Transfection, Control