primary antibodies against lamp1  (Developmental Studies Hybridoma Bank)

Journal: bioRxiv

Article Title: Mechanoresilience of lysosomes conferred by TMEM63A

doi: 10.64898/2025.12.18.695245

Figure Lengend Snippet: A) LysoFlipper FLIM in WT RAW264.7 macrophages incubated with 1 mM LLOMe for 5 or 15 min, or 250 µM GPN for 15 min. Each dot represents one field of 5-10 cells. N = 3. B) qPCR for TMEM63A, B, and C in RAW264.7, BMDM, or MEF. n = 3. C-D) TMEM63A-OFP (magenta) and LAMP1-GFP (green) in WT RAW264.7 cells before and after challenge with IgG-coated microspheres. E-F) 10 kDa dextran or SRG pulsed for 16 h and chased for 1 h in WT and TMEM63A KO BMDM. G) Lysosomal pH in WT and TMEM63A KO BMDM determined using ratiometric measurements of 10 kDa Oregon Green dextran pulsed for 16 h and chased for 1 h. Each data point represents a field containing >5 cells. n = 3. H) LAMP1, cathepsin C expression and processing by WB. I) DQ-BSA signal of WT and TMEM63A KO BMDM after pulse (1 h) and chase (1 h). All data points represent a field containing 4-8 cells. n = 3. J-K) Phagocytosis in WT and TMEM63A KO BMDM, quantified in K. Each data point represents 3-5 fields containing 15-25 cells. n = 3, scale bars 10 µm.

Article Snippet: Primary antibodies against LAMP1 (DSHB, 1D4B), Cathepsin C (Santa Cruz, sc-74590) and GAPDH (Santa Cruz, 365062) were used at 1:1000 (v/v) for western blotting.

Techniques: Incubation, Expressing

Journal: bioRxiv

Article Title: Phagosome Maturation in Macrophages is Enhanced by p38α MAPK Signaling

doi: 10.1101/2025.07.03.662985

Figure Lengend Snippet: A – Schematic describing the design of experiment prior to microscopy. B – Representative maximum intensity projections (MIPs) of RAW 264.7 cells at indicated time points after introduction of particles. Magenta – LAMP1, Green – 500 nm-sized non-modified polystyrene particles, Blue – Hoechst. Scale bar – 10 μm. C – Percentage of internalised particles per cell that colocalize with LAMP1 signal at indicated time points. Kruskal-Wallis test, N ≥ 3 independent experiments, 116 cells per group on average. Data represented as violin plots with colored lines indicating mean, black lines indicating median and dashed lines indicating quartiles of the data. D – Representative MIPs of RAW 264.7 cells stained with LysoTracker Red (LTR) at indicated time points after introduction of particles. Magenta – LTR, Green – 500 nm-sized non-modified polystyrene particles. Scale bar – 10 μm. E – Percentage of internalised particles per cell that colocalize with LTR signal at indicated time points, as obtained using the PCC-based method for analysis of colocalisation. Kruskal-Wallis test, N = 3 independent experiments, 55 cells per group on average. Data represented as violin plots with colored lines indicating mean, dark lines indicating median and dotted lines indicating quartiles of the data. F – Representative MIPs of RAW 264.7 cells at indicated times post infection with E. coli (MOI = 10:1). Magenta – LTR, Green – EGFP-expressing E. coli. Scale bar – 5 μm. G – Percentage of bacteria taken up by a cell that were localized in lysosomes. Kruskal-Wallis test, N = 3 independent experiments, n = 47 cells per group on average. Bars indicate the arithmetic mean of the data.

Article Snippet: Primary antibody against LAMP1 (rat anti-mouse) was procured from Developmental Studies Hybridoma Bank, Iowa (Clone number: 1D4B) and primary and secondary antibodies used for Western blot assays (rabbit anti-mouse: p38 MAPK – polyclonal, GAPDH – clone 14C10 and β tubulin – polyclonal, and goat anti-rabbit: HRP-linked anti-rabbit IgG) were purchased from Cell Signaling Technologies, USA.

Techniques: Microscopy, Modification, Staining, Infection, Expressing, Bacteria

Journal: bioRxiv

Article Title: Phagosome Maturation in Macrophages is Enhanced by p38α MAPK Signaling

doi: 10.1101/2025.07.03.662985

Figure Lengend Snippet: A – Representative MIPs of untreated cells (control) or LPS-activated RAW 264.7 cells at indicated time points post uptake of 500 nm non-modified polystyrene particles (Green) and stained with LTR (Magenta). Scale bar – 10 μm. B – Percentage colocalization of particles with LTR signal at indicated time points. Mann Whitney test; N = 3 independent experiments, 49 cells per group on average. C – Representative MIPs of primary peritoneal macrophages isolated from mice and either left untreated (control) or activated with LPS before ex vivo phagocytosis of particles. Magenta – LAMP1; Green – 500 nm-sized polystyrene particles. Scale bar – 10 μm. D – Percentage of internalized particles per cell that colocalize with LAMP1 in mouse peritoneal macrophages. Mann Whitney test; N = 3 independent experiments, 66 cells per group on average. E – Representative MIPs of RAW 264.7 cells after uptake of either control or LPS-adsorbed 500 nm-sized fluorescent particles at the indicated time points. Magenta – LysoTracker Red, Green – 500nm sized particles. Scale bars – 10 μm. F – Percentage colocalization of particles with LTR signal at indicated time points. Mann-Whitney test; N = 3 independent experiments, 94 cells per group on average. G – Representative MIPs of primary peritoneal macrophages isolated from mice after in vivo phagocytosis of particles. Magenta – LAMP1; Green – 500 nm sized particles Scale bars – 10 μm. H – Percentage of internalized particles per cell that colocalize with LAMP1 in mouse peritoneal macrophages. Mann Whitney test; N = 4 mice (control) and 3 mice (LPSads), 144 and 103 cells per group respectively. In all violin plots, dark lines represent median, and dashed lines represent quartiles of the data.

Article Snippet: Primary antibody against LAMP1 (rat anti-mouse) was procured from Developmental Studies Hybridoma Bank, Iowa (Clone number: 1D4B) and primary and secondary antibodies used for Western blot assays (rabbit anti-mouse: p38 MAPK – polyclonal, GAPDH – clone 14C10 and β tubulin – polyclonal, and goat anti-rabbit: HRP-linked anti-rabbit IgG) were purchased from Cell Signaling Technologies, USA.

Techniques: Control, Modification, Staining, MANN-WHITNEY, Isolation, Ex Vivo, In Vivo

Journal: bioRxiv

Article Title: Phagosome Maturation in Macrophages is Enhanced by p38α MAPK Signaling

doi: 10.1101/2025.07.03.662985

Figure Lengend Snippet: A – Representative MIPs of RAW 264.7 cells 2 hours after uptake of either control particles or particles conjugated with IgG, albumin or folic acid. Magenta – LAMP1, Green – particles. Scale bars – 5 μm. B – Percentage of phagocytosed particles per cell that colocalize with LAMP1 signal in RAW264.7 cells. Kruskal-Wallis test; N = 3 independent experiments, 130 cells per group on average. C – Representative MIPs of primary peritoneal macrophages isolated from mice 2h after ex vivo internalization of either control particles or particles conjugated with either mouse IgG, albumin or folic acid. Magenta – LAMP1; Green – 500 nm-sized polystyrene particles. Scale bars – 10 μm. D – Percentage of phagocytosed particles per cell that colocalize with LAMP1 signal in mouse peritoneal macrophages. Kruskal-Wallis test; N = 3 independent experiments, 58 cells per group on average. In all violin plots, dark lines represent median and dashed lines represent quartiles of the data.

Article Snippet: Primary antibody against LAMP1 (rat anti-mouse) was procured from Developmental Studies Hybridoma Bank, Iowa (Clone number: 1D4B) and primary and secondary antibodies used for Western blot assays (rabbit anti-mouse: p38 MAPK – polyclonal, GAPDH – clone 14C10 and β tubulin – polyclonal, and goat anti-rabbit: HRP-linked anti-rabbit IgG) were purchased from Cell Signaling Technologies, USA.

Techniques: Control, Isolation, Ex Vivo

Journal: bioRxiv

Article Title: Phagosome Maturation in Macrophages is Enhanced by p38α MAPK Signaling

doi: 10.1101/2025.07.03.662985

Figure Lengend Snippet: A – Representative MIPs of RAW 264.7 cells 2 hours post uptake of particles. Cells were treated with p38MAPK inhibitor SB203580 (10 μM) or vehicle (DMSO) for 1h followed by 100 ng/mL LPS for 18h before uptake, or left untreated (control). Magenta – LAMP1, Green – 500 nm polystyrene particles. Scale bars – 10 μm. B – Percentage of particles taken up per cell that colocalize with LAMP1 signal 2 hours post uptake in RAW 264.7 cells after treatments described in A. Kruskal-Wallis (with Dunn’s post-hoc) test; N = 3 independent experiments, n = 219 cells per group on average. C – Representative MIPs of RAW 264.7 cells which were treated with either 10 μM SB203580 or vehicle (DMSO) or received no treatment before internalization of either non-modified (control) or LPS ads particles (all other groups). Treatments occurred 2 hours-post addition of particles. Magenta – LAMP1, Green – 500 nm polystyrene particles (either non-modified – control or LPS adsorbed). Scale bars – 10 μm. D – Percentage of particles taken up per cell that localize to lysosomes, 2h post uptake after treatments described in C. Kruskal-Wallis (with Dunn’s post-hoc) test; N ≥ 3 independent experiments, n = 226 cells on average per group. Data are plotted as violin plots with black lines indicating median, dashed lines indicating quartiles, and colored lines indicating mean of the data.

Article Snippet: Primary antibody against LAMP1 (rat anti-mouse) was procured from Developmental Studies Hybridoma Bank, Iowa (Clone number: 1D4B) and primary and secondary antibodies used for Western blot assays (rabbit anti-mouse: p38 MAPK – polyclonal, GAPDH – clone 14C10 and β tubulin – polyclonal, and goat anti-rabbit: HRP-linked anti-rabbit IgG) were purchased from Cell Signaling Technologies, USA.

Techniques: Control, Modification

Journal: Redox Biology

Article Title: Schaftoside improves HFpEF through regulation the autophagy-lysosome pathway by allosterically targeting CaMKII-δ

doi: 10.1016/j.redox.2024.103424

Figure Lengend Snippet: Schaftoside enhanced lysosome autophagy. (A) TEM images showed that SS promoted the formation of autolysosome in myocardial tissue. The down panels showed the enlarged images of selected areas defined by red boxes in the up panels. Yellow arrow: autolysosomes. Quantification of autolysosomes in TEM images (3–4 samples per group, n = 6 images). (B) Co-staining of heart sections with cTnl (red)，Lamp1 antibody (green) and nuclei (DAPI, stain, blue) by immunofluorescence ( n = 6). (C) Lamp1, P62 and LC3 protein levels in myocardial tissue from mice of different groups tested by Western blot analysis ( n = 3). (D) TEM images showing that SS (80 ng/mL) promoted the formation of autophagosome in NMCM cells; The down panels show the enlarged images of selected areas defined by red boxes in the left panels. Yellow arrow: lysosomes. Quantification of autolysosomes in TEM images (3 samples per group, n = 5 images). (E) Immunofluorescence images showed that SS improved lysosomal function inhibited by AngII in NMCM cells. Lamp1 antibodies (green) and nucleus (DAPI, blue) ( n = 3). (F) Lamp1 protein levels in NMCM cells from SS tested by Western blot analysis ( n = 4). (G, H) Determination of lysosomal pH in cardiomyocytes using the ratiometric dye LysoSensor Yellow/Blue DND-160, followed by quantification of fluorescence signals. Acidic organelles predominantly exhibit yellow fluorescence (pH∼4.5), while less acidic organelles display more blue fluorescence (pH∼6) ( n = 3). (I, J) Cells were transfected with mRFP-GFP-LC3 adenovirus for 48 h. Autophagosomes are expressed as yellow puncta, and autolysosomes are expressed as red puncta ( n = 4); Scale bar = 50 μm. (K) Baf-A1 (100 nM), which disrupted autophagosome-lysosome fusion and the acidification of autolysosomes, inhibited the reduction of LC3II levels triggered by SS in cardiomyocytes. All graphs show mean ± SEM. Statistical analysis was performed using ordinary-one way ANOVA. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Next, the membranes were blocked with 5 % skimmed milk for 1 h. They were probed with primary antibodies against CaMKII-δ, p -CaMKII-δ and Lamp1 (Invitrogen, CA, USA), P62 and LC3 (CST, MA, USA), AKT and p -AKT (Abclonal, Wuhan, China), diluted at 1:1000 in primary antibody dilution buffer at 4 °C overnight.

Techniques: Staining, Immunofluorescence, Western Blot, Fluorescence, Transfection

Journal: Redox Biology

Article Title: Schaftoside improves HFpEF through regulation the autophagy-lysosome pathway by allosterically targeting CaMKII-δ

doi: 10.1016/j.redox.2024.103424

Figure Lengend Snippet: Schaftoside improves lysosomal autophagy by targeting CaMKII-δ. (A) Lamp1 protein levels in cells from SS tested by Western blot analysis ( n = 4). (B) NMCMs were treated with adenovirus (Ad) to overexpress CaMKII-δ, and changes in lysosomal activity (LysoSensor Yellow/Blue staining) were investigated ( n = 3). (C) Immunofluorescence for Lamp1 (green) and the indicated nucleus markers (blue) ( n = 3). (D, E) NMCMs were transfected with mRFP-GFP-LC3 adenovirus for 48 h. Autophagosomes were expressed as yellow puncta and autolysosomes were expressed as red puncta ( n = 3). Scale bars: 50 μm. All graphs show mean ± SEM. Statistical analysis was performed using ordinary-one way ANOVA. ∗ P < 0.05，∗∗ P < 0.01，∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Next, the membranes were blocked with 5 % skimmed milk for 1 h. They were probed with primary antibodies against CaMKII-δ, p -CaMKII-δ and Lamp1 (Invitrogen, CA, USA), P62 and LC3 (CST, MA, USA), AKT and p -AKT (Abclonal, Wuhan, China), diluted at 1:1000 in primary antibody dilution buffer at 4 °C overnight.

Techniques: Western Blot, Activity Assay, Staining, Immunofluorescence, Transfection