anti lamp1 antibody  (Bioss)

Journal: EMBO Molecular Medicine

Article Title: MHC-I upregulation by macbecin II in the solid tumors potentiates the effect of active immunotherapy

doi: 10.1038/s44321-025-00213-7

Figure Lengend Snippet: ( A ) Flow cytometry analysis for INF-γ-CD8 + T cells in the four groups. ( B ) Granzyme B expression was examined by immunohistochemistry (left) and quantified (right) in the four groups. The staining intensity was analyzed by the unpaired two-tailed Student’s t-test ( n = 4/group, biological replicates). ( C ) LAMP1 expression was examined through immunohistochemistry (left) and quantified (right) in the four groups. The staining intensity was measured and analyzed by the one-way ANOVA with a Tukey post-hoc test ( n = 4/group, biological replicates). ( D , E ) MHC-I expression was examined in EPCAM+ tumor cells and CD11c+ dendritic cells in the dissociated tumor by FACS. Statistical analysis was performed using the one-way ANOVA with a Tukey post-hoc test ( n = 3/group, biological replicates). ( F , G ) Animal weight ( F ) and AST activity ( G ) in the blood of mice were measured at the end-point. Data are represented as mean +/− SEM ( n = 5/group, biological replicates). ( H ) B16-Ova cells (500 cells/well, n = 3/group, biological replicates, were seeded in a 96-well plate and treated with a combination of macbecin II (0.1 µM) and OT-1 T cells (E:T ratio 5:1) with or without ectopic MHC-I expression for 48 h. The T cells were activated with IL2-ep13nsEV that were isolated from B16-Ova lysate-pulsed BMDCs (E:T ratio 5:1). After the incubation, cells were washed with ice-cold PBS to remove dead cells. The live cells were fixed with methanol at room temperature for 15 min and stained with crystal violet. The dye was dissolved in 10% acetic acid and measured at 590 nm. ( I ) E0771 cells (500 cells/well, n = 3/group, biological replicates) were seeded in a 96-well plate and treated with a combination of T cells (E:T ratio 5:1) and macbecin II (0.1 µM) with or without ectopic MHC-I expression for 48 h. The T cells were isolated from the spleen of E0771-bearing syngeneic mouse and they were activated with IL2-ep13nsEV that were isolated from E0771 lysate-pulsed BMDCs. After the incubation, cells were processed as described in ( H ). Statistical significance was determined by the one-way ANOVA with a Tukey post-hoc test. ( J ) MHC-I was ectopically expressed in B16-F1 cells, and the MHC-I expression was confirmed by FACS. The result was analyzed by the unpaired two-tailed Student’s t-test ( n = 3/group, biological replicates). ( K ) Cell viability was examined by MTS assay for B16-F1 with or without MHC-I over expression (OE) ( n = 3/group, biological replicates). ( L , M ) Animal weight ( L ) and AST activity ( M ) were measured at the end-point ( n = 5/group, biological replicates). Data are presented as mean +/− SEM.

Article Snippet: LAMP1 antibody , Bioss , BS-1970R.

Techniques: Flow Cytometry, Expressing, Immunohistochemistry, Staining, Two Tailed Test, Activity Assay, Isolation, Incubation, MTS Assay, Over Expression

Journal: EMBO Molecular Medicine

Article Title: MHC-I upregulation by macbecin II in the solid tumors potentiates the effect of active immunotherapy

doi: 10.1038/s44321-025-00213-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: LAMP1 antibody , Bioss , BS-1970R.

Techniques: Recombinant, Over Expression, Plasmid Preparation, Sequencing, Proliferation Assay, Red Blood Cell Lysis, Staining, Activation Assay, Isolation, Cell Isolation, cDNA Synthesis, Bradford Protein Assay, AST Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: EMBO Molecular Medicine

Article Title: MHC-I upregulation by macbecin II in the solid tumors potentiates the effect of active immunotherapy

doi: 10.1038/s44321-025-00213-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: LAMP1 antibody , Bioss , BS-1970R.

Techniques: Recombinant, Over Expression, Plasmid Preparation, Sequencing, Proliferation Assay, Red Blood Cell Lysis, Staining, Activation Assay, Isolation, Cell Isolation, cDNA Synthesis, Bradford Protein Assay, AST Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Fish & shellfish immunology

Article Title: Neu1-Deficient Zebrafish Cells Exhibit Reduced Edwardsiella piscicida Infection Due to Altered Lysosomal Exocytosis and Membrane Dynamics.

doi: 10.1016/j.fsi.2025.110273

Figure Lengend Snippet: Fig. 1. Properties of zebrafish primary cells originating from fins. Primary cultured cells were prepared from WT and Neu1-KO zebrafish fins. (A) Morphology of the zebrafish fin cells. (B) Sialidase activity. n = 3. (C) The protein levels of Lamp1 and β-Actin were analyzed by immunoblotting with the cell lysate. (D) Quantitative analysis of the intensities of Lamp1 and β-Actin bands in (C) were carried out and the results are presented as relative Lamp1/β-Actin level to the value in WT cells. n = 4 for each group. (E) Distribution of the Lamp1 protein in cultured fin cells. Lamp1 (red), actin filaments (green), and nuclei (blue) were stained and observed using a fluorescence microscope. The white rectangle shown in the third panel is magnified as the fourth panel. White bar means the scale of 20 μm. White arrows indicate Lamp1 signals in the plasma membrane. Results are shown as means ± standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: EGFR, Lamp1, Gapdh, and β-Actin were detected by incubation with rabbit polyclonal antiEGFR antibody (1/1000 dilution, SC-03, Santa Cruz Biotechnoloy, TX, USA), rabbit polyclonal anti-Lamp1 antibody (1/1000, ab24170, Abcam, Cambridge, UK), rabbit polyclonal anti GAPDH antibody (1/ 1000, 60004-1-Ig, Proteintech, IL, USA), and mouse monoclonal antiβ-Actin antibody (1/1000, 66009-1-Ig, Proteintech), respectively, followed by reaction with secondary HRP-anti-mouse or anti-rabbit IgG antibody (1/10,000).

Techniques: Cell Culture, Activity Assay, Western Blot, Staining, Fluorescence, Microscopy, Clinical Proteomics, Membrane, Standard Deviation

Journal: Fish & shellfish immunology

Article Title: Neu1-Deficient Zebrafish Cells Exhibit Reduced Edwardsiella piscicida Infection Due to Altered Lysosomal Exocytosis and Membrane Dynamics.

doi: 10.1016/j.fsi.2025.110273

Figure Lengend Snippet: Fig. 2. Suppression of E. piscicida infection in Neu1-KO cells via the enhanced lysosomal exocytosis. (A) E. piscicida infection in zebrafish primary cells. Results were shown as ratio to colony number in WT cells. n = 10. (B) The protein levels of Lamp1 and Gapdh were analyzed by immunoblotting with the cell lysate with E. piscicida infection. (C) Quantitative analysis of the intensities of Lamp and Gapdh bands in (B) were carried out and the results are presented as relative Lamp1/Gapdh level to the value in WT cells. n = 3 for each group. (D) Distribution of Lamp1 protein in the cultured Neu1-KO cells. Lamp1 (red), actin filament (green), and nucleus (blue) were stained and observed by fluorescence microscopy. White bar means the scale of 20 μm. White arrows indicate the Lamp1 signals at the plasma membrane. (E) E. piscicida infection in Neu1-KO cells with BAPTA-AM pretreatment. Results were shown as ratio to colony number in vehicle (DMSO). n = 10. Results were shown as means ± standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: EGFR, Lamp1, Gapdh, and β-Actin were detected by incubation with rabbit polyclonal antiEGFR antibody (1/1000 dilution, SC-03, Santa Cruz Biotechnoloy, TX, USA), rabbit polyclonal anti-Lamp1 antibody (1/1000, ab24170, Abcam, Cambridge, UK), rabbit polyclonal anti GAPDH antibody (1/ 1000, 60004-1-Ig, Proteintech, IL, USA), and mouse monoclonal antiβ-Actin antibody (1/1000, 66009-1-Ig, Proteintech), respectively, followed by reaction with secondary HRP-anti-mouse or anti-rabbit IgG antibody (1/10,000).

Techniques: Infection, Western Blot, Cell Culture, Staining, Fluorescence, Microscopy, Clinical Proteomics, Membrane, Standard Deviation

Journal: Journal of Neuroinflammation

Article Title: Glutamine metabolism modulates microglial NLRP3 inflammasome activity through mitophagy in Alzheimer’s disease

doi: 10.1186/s12974-024-03254-w

Figure Lengend Snippet: Reduced glutamine metabolism enhances microglial mitophagy and inhibits NLRP3 inflammasome activation. ( A ) Western blots of pro- and mature IL-1β and caspase-1 in lysates and supernatants of LoAMs in the presence or absence of the anti-oxidant and free radical scavenger NAC ( n = 3). ND, not detected. ( B ) Western blots and densitometry quantification of the mitophagy protein PINK1, parkin, and p62 in LoAMs in the presence or absence of BPTES ( n = 3). ( C ) Autophagosome formation indicated by puncta and quantitated by confocal microscopy in LoAMs in the presence or absence of BPTES or GLS1 siRNA. Scale bar: 10 μm ( n = 6). ( D ) Confocal microscopy detection and quantitation of co-localization of the mitochondrial protein TOM20 (green), lysosomal protein LAMP1 (red), and DAPI (blue) in LoAMs in the presence or absence of BPTES or GLS1 siRNA. Scale bar: 10 μm ( n = 6). ( E ) Confocal microscopy detection and quantitation of co-staining of MitoTracker ang LysoTracker in LoAMs in the presence or absence of BPTES or GLS1 siRNA. Scale bar: 10 μm ( n = 6). ( F ) WB analysis of pro- and mature IL-1β and caspase-1 in lysates and supernatants of LoAMs in the presence or absence of mitophagy stimulator NMN ( n = 3). n represents the number of biological replicates. Data are presented as the mean ± SEM. Statistical significance was determined by one-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: After fixed with 4% PFA, permeabilized using 0.5% triton X-100 and blocked in 5% BSA, the coverslips were then stained with mouse monoclonal anti-TOM20 (1:200; 66777-1-Ig, Proteintech) and rabbit polyclonal anti-LAMP1 (1:100; bs-1970R, Bioss) or goat polyclonal anti-ASC (1:50; ab175449, Abcam) and rabbit polyclonal anti-NLRP3 (1:500; 19771-1-AP, Proteintech) overnight at 4℃.

Techniques: Activation Assay, Western Blot, Confocal Microscopy, Quantitation Assay, Staining

Journal: Journal of Neuroinflammation

Article Title: Glutamine metabolism modulates microglial NLRP3 inflammasome activity through mitophagy in Alzheimer’s disease

doi: 10.1186/s12974-024-03254-w

Figure Lengend Snippet: Reduced glutamine metabolism enhances mitophagy in LPS-primed oAβ 1−42 -treated primary microglia via AMPK/mTORC1 signaling. (A and B) WB analysis and densitometry-based quantification of raptor and p-AMPK in LoAMs in the presence or absence of BPTES ( A ) and GLS1 siRNA ( B ) ( n = 3). ( C ) Autophagosome formation indicated by puncta and quantitated by confocal microscopy in LoAMs with or without rapamycin. Scale bar: 10 μm ( n = 6). ( D ) Confocal microscopy detection and quantitation of co-localization of the mitochondrial protein TOM20 (green), lysosomal protein LAMP1 (red), and DAPI (blue) in LoAMs in the presence or absence of rapamycin. Scale bar: 10 μm ( n = 6). ( E ) Confocal microscopy detection and quantitation of co-staining of MitoTracker ang LysoTracker in LoAMs in the presence or absence of rapamycin. Scale bar: 10 μm ( n = 6). ( F ) Western blot analysis of pro- and mature IL-1β and caspase-1 in lysates and supernatants of LoAMs in the presence or absence of rapamycin ( n = 3). ND, not detected. n represents the number of biological replicates. Data are presented as the mean ± SEM. Statistical significance was determined by one-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; NS, not significant

Article Snippet: After fixed with 4% PFA, permeabilized using 0.5% triton X-100 and blocked in 5% BSA, the coverslips were then stained with mouse monoclonal anti-TOM20 (1:200; 66777-1-Ig, Proteintech) and rabbit polyclonal anti-LAMP1 (1:100; bs-1970R, Bioss) or goat polyclonal anti-ASC (1:50; ab175449, Abcam) and rabbit polyclonal anti-NLRP3 (1:500; 19771-1-AP, Proteintech) overnight at 4℃.

Techniques: Confocal Microscopy, Quantitation Assay, Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Transcranial Magneto-Acoustic Stimulation Protects Synaptic Rehabilitation from Amyloid-Beta Plaques via Regulation of Microglial Functions

doi: 10.3390/ijms25094651

Figure Lengend Snippet: TUS and TMAS treatments attenuated amyloid burdens and amyloid-induced neurotoxicity. ( A ) Coronal sections of sham-, TUS-, or TMAS-treated 5xFAD mice were stained with DAPI (blue) for nuclei, anti-Aβ (red) for Aβ plaques, and Iba1 (green) for microglia. Representative images of the hippocampal regions are shown. Original magnification ×20; scale bar: 100 μm. ( B ) Statistical analysis of the difference in the Aβ fluorescence areas between sham, TUS, and TMAS groups (6 mice from each group were used for analysis). ( C ) Quantitation of the number of plaque-associated microglia in A (6 mice from each group were used for analysis). ( D ) Quantitation of the area of Lamp1-positive dystrophic neurites in E (6 mice from each group were used for analysis). ( E ) Coronal sections from sham-, TUS-, or TMAS-treated 5xFAD mice were stained with DAPI (blue) for nuclei, anti-Aβ (red) for Aβ, and Lamp1 (green) for dystrophic neurites. Representative images of the hippocampus regions are shown. Original magnification ×40; scale bar: 50 μm. Zoom-in images on the right have a scale bar equal to 20 μm. All data in the results are expressed as mean ± SEM. * p < 0.05; *** p < 0.001.

Article Snippet: The primary antibodies used were as follows: anti-Iba1 (Wako; Cat# 016-20001, RRID: AB_839506, 1:500, Osaka, Japan), anti-Iba1 (Santa Cruz Biotechnology; Cat# sc-32725, RRID: AB_667733, 1:500, Santa Cruz, CA, USA), anti-Aβ (Abcam; Cat# ab32136, RRID: AB_2289606, 1/500, Cambridge, UK), anti-PSD95 (Cell Signaling Technology; Cat# 3450S, RRID: AB_2292883, 1:2000, Danvers, MA, USA), anti-CD11c (Biolegend; Cat# 117304, RRID: AB_313773, 1:1000, Santa Cruz, CA, USA), anti-Lamp1 (BIOSS; Cat# bs-1970R, 1:100, Beijing, China), and anti-CD68 (Bio-Rad; Cat# MCA1957, RRID: AB_322219, 1:50, Heraklion, CA, USA).

Techniques: Staining, Fluorescence, Quantitation Assay