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mouse antibody to lamp1 1d4b  (Developmental Studies Hybridoma Bank)


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    Developmental Studies Hybridoma Bank mouse antibody to lamp1 1d4b
    ARF GTPases are recruited to damaged lysosomes. (A) HeLa cells were transfected with HA-tagged ARF1, ARF5, or ARF6 and treated the next day with 0.5 mM LLOME for 1 h or 3 h. At each time point, cells were fixed and stained for HA (cyan) and endogenous <t>LAMP1</t> (magenta) and imaged with super-resolution confocal microscopy (Nikon NSPARC system). Scale bar indicates 10 µm for original size images and 1 µm for zoomed images. (B) Quantification of data in (A) n = 15 cells. Error bars represent mean +SD. Data were analyzed with one-way ANOVA. *p < 0.05 (C) (left) HEK293 cells stably expressing 3XHA-tagged TMEM192 were transfected with GFP-tagged ARF1, ARF5, or ARF6 constructs. Untreated and LLOME-treated (3 h) cells were subjected to lysosome immunoprecipitation using HA-conjugated magnetic beads (LysoIP). Total cell lysates and immunoprecipitates were immunoblotted for GFP to detect bound ARFs (left). Lysates and immunoprecipitates were also immunoblotted for endogenous LAMP1, Golgin-97, or EEA1 (right). (D) Quantification of data in (C) . Bars indicate mean +SD of ≥2 experimental replicates. Statistical analysis was performed using unpaired t-test. *p < 0.05. Statistics represent comparison of untreated HEK293 cells with 3-h LLOME (1 mM) treated cells quantified by densitometry. (E) HEK293 cells were treated with LLOME or not and subjected to the LysoIP protocol. Lysates were immunoblotted and stained for endogenous ARF5 and HA. (F) Quantification of data in (E) . Bars indicate mean +SD of three experimental replicates. *p < 0.05. Statistics represent comparison of untreated HEK293 cells with 20-min and 3-h LLOME-treated cells quantified by densitometry.
    Mouse Antibody To Lamp1 1d4b, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 99/100, based on 2329 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Loss of ARF5 impairs recovery after lysosomal damage"

    Article Title: Loss of ARF5 impairs recovery after lysosomal damage

    Journal: Frontiers in Molecular Biosciences

    doi: 10.3389/fmolb.2025.1699266

    ARF GTPases are recruited to damaged lysosomes. (A) HeLa cells were transfected with HA-tagged ARF1, ARF5, or ARF6 and treated the next day with 0.5 mM LLOME for 1 h or 3 h. At each time point, cells were fixed and stained for HA (cyan) and endogenous LAMP1 (magenta) and imaged with super-resolution confocal microscopy (Nikon NSPARC system). Scale bar indicates 10 µm for original size images and 1 µm for zoomed images. (B) Quantification of data in (A) n = 15 cells. Error bars represent mean +SD. Data were analyzed with one-way ANOVA. *p < 0.05 (C) (left) HEK293 cells stably expressing 3XHA-tagged TMEM192 were transfected with GFP-tagged ARF1, ARF5, or ARF6 constructs. Untreated and LLOME-treated (3 h) cells were subjected to lysosome immunoprecipitation using HA-conjugated magnetic beads (LysoIP). Total cell lysates and immunoprecipitates were immunoblotted for GFP to detect bound ARFs (left). Lysates and immunoprecipitates were also immunoblotted for endogenous LAMP1, Golgin-97, or EEA1 (right). (D) Quantification of data in (C) . Bars indicate mean +SD of ≥2 experimental replicates. Statistical analysis was performed using unpaired t-test. *p < 0.05. Statistics represent comparison of untreated HEK293 cells with 3-h LLOME (1 mM) treated cells quantified by densitometry. (E) HEK293 cells were treated with LLOME or not and subjected to the LysoIP protocol. Lysates were immunoblotted and stained for endogenous ARF5 and HA. (F) Quantification of data in (E) . Bars indicate mean +SD of three experimental replicates. *p < 0.05. Statistics represent comparison of untreated HEK293 cells with 20-min and 3-h LLOME-treated cells quantified by densitometry.
    Figure Legend Snippet: ARF GTPases are recruited to damaged lysosomes. (A) HeLa cells were transfected with HA-tagged ARF1, ARF5, or ARF6 and treated the next day with 0.5 mM LLOME for 1 h or 3 h. At each time point, cells were fixed and stained for HA (cyan) and endogenous LAMP1 (magenta) and imaged with super-resolution confocal microscopy (Nikon NSPARC system). Scale bar indicates 10 µm for original size images and 1 µm for zoomed images. (B) Quantification of data in (A) n = 15 cells. Error bars represent mean +SD. Data were analyzed with one-way ANOVA. *p < 0.05 (C) (left) HEK293 cells stably expressing 3XHA-tagged TMEM192 were transfected with GFP-tagged ARF1, ARF5, or ARF6 constructs. Untreated and LLOME-treated (3 h) cells were subjected to lysosome immunoprecipitation using HA-conjugated magnetic beads (LysoIP). Total cell lysates and immunoprecipitates were immunoblotted for GFP to detect bound ARFs (left). Lysates and immunoprecipitates were also immunoblotted for endogenous LAMP1, Golgin-97, or EEA1 (right). (D) Quantification of data in (C) . Bars indicate mean +SD of ≥2 experimental replicates. Statistical analysis was performed using unpaired t-test. *p < 0.05. Statistics represent comparison of untreated HEK293 cells with 3-h LLOME (1 mM) treated cells quantified by densitometry. (E) HEK293 cells were treated with LLOME or not and subjected to the LysoIP protocol. Lysates were immunoblotted and stained for endogenous ARF5 and HA. (F) Quantification of data in (E) . Bars indicate mean +SD of three experimental replicates. *p < 0.05. Statistics represent comparison of untreated HEK293 cells with 20-min and 3-h LLOME-treated cells quantified by densitometry.

    Techniques Used: Transfection, Staining, Confocal Microscopy, Stable Transfection, Expressing, Construct, Immunoprecipitation, Magnetic Beads, Comparison

    Lipid transfer proteins OSBP and ORP9 are recruited to damaged lysosomes in an ARF-independent manner. (A) HeLa cells were transfected with empty vector (shCTRL) or pLKO containing ARF1 or ARF5 shRNAs. Cells were treated or not with LLOME (0.5 mM) for 35 min, fixed and stained for endogenous OSBP (green), LAMP1 (red), and TGN46 (magenta), and imaged with confocal microscopy. Scale bars indicate 10 µm. (B) HEK293 cells stably expressing tagged TMEM192 were transfected with empty pLKO vector (shCTRL) or pLKO containing ARF1 or ARF5 shRNAs. Untreated and LLOME (1 mM)-treated cells were subjected to the LysoIP protocol. Precipitated lysosomes were immunoblotted for endogenous OSBP. (C) HeLa cells were transfected with empty vector (shCTRL), ARF5, or ARF6 shRNAs. Cells were treated or not with LLOME (0.5 mM) for 20 min, fixed and stained for ORP9 (green), LAMP1 (red), and F-actin (blue), and imaged with confocal microscopy. Scale bars indicate 10 µm. (D) Quantification of data in (C) n = 28 cells. Error bars represent mean +SD. Data were analyzed with two-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance. *p < 0.05.
    Figure Legend Snippet: Lipid transfer proteins OSBP and ORP9 are recruited to damaged lysosomes in an ARF-independent manner. (A) HeLa cells were transfected with empty vector (shCTRL) or pLKO containing ARF1 or ARF5 shRNAs. Cells were treated or not with LLOME (0.5 mM) for 35 min, fixed and stained for endogenous OSBP (green), LAMP1 (red), and TGN46 (magenta), and imaged with confocal microscopy. Scale bars indicate 10 µm. (B) HEK293 cells stably expressing tagged TMEM192 were transfected with empty pLKO vector (shCTRL) or pLKO containing ARF1 or ARF5 shRNAs. Untreated and LLOME (1 mM)-treated cells were subjected to the LysoIP protocol. Precipitated lysosomes were immunoblotted for endogenous OSBP. (C) HeLa cells were transfected with empty vector (shCTRL), ARF5, or ARF6 shRNAs. Cells were treated or not with LLOME (0.5 mM) for 20 min, fixed and stained for ORP9 (green), LAMP1 (red), and F-actin (blue), and imaged with confocal microscopy. Scale bars indicate 10 µm. (D) Quantification of data in (C) n = 28 cells. Error bars represent mean +SD. Data were analyzed with two-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance. *p < 0.05.

    Techniques Used: Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Stable Transfection, Expressing

    ARF5-depleted cells do not recover from lysosomal damage. (A) Schematic of Gal3 recovery assay. (B) HeLa cells were transfected with empty vector control (shCTRL), ARF5, or ARF6 shRNAs. Cells were treated or not with LLOME (0.5 mM) which was rinsed out after 20 min of incubation. Cells were allowed to recover for either 1 h or 8 h, after which they were fixed and stained for endogenous Gal3 (cyan), LAMP1 (magenta), and F-actin (yellow) and imaged with confocal microscopy. Scale bar indicates 10 µm. (C) Quantification of data in (B) n = 40–66 cells from two independent experiments. Error bars represent mean +SD. Data were analyzed with nonparametric Kruskal–Wallis one-way ANOVA test with Dunn’s correction. *p < 0.05 (D) HeLa cells were transfected with empty vector (shCTRL), ARF5, or ARF6 shRNAs and plated in black-walled 96-well plates. Cells were then treated with LLOME overnight or after 20 min of incubation, rinsed and allowed to recover (LLOME + recovery). Cells were incubated overnight, fixed the next day, and stained for actin (Alexa-488 phalloidin). Fluorescence was analyzed using a BioTek imaging microplate reader. y-axis represents Alexa-488 nm fluorescence [AU]. Data were analyzed with two-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance. *p < 0.05.
    Figure Legend Snippet: ARF5-depleted cells do not recover from lysosomal damage. (A) Schematic of Gal3 recovery assay. (B) HeLa cells were transfected with empty vector control (shCTRL), ARF5, or ARF6 shRNAs. Cells were treated or not with LLOME (0.5 mM) which was rinsed out after 20 min of incubation. Cells were allowed to recover for either 1 h or 8 h, after which they were fixed and stained for endogenous Gal3 (cyan), LAMP1 (magenta), and F-actin (yellow) and imaged with confocal microscopy. Scale bar indicates 10 µm. (C) Quantification of data in (B) n = 40–66 cells from two independent experiments. Error bars represent mean +SD. Data were analyzed with nonparametric Kruskal–Wallis one-way ANOVA test with Dunn’s correction. *p < 0.05 (D) HeLa cells were transfected with empty vector (shCTRL), ARF5, or ARF6 shRNAs and plated in black-walled 96-well plates. Cells were then treated with LLOME overnight or after 20 min of incubation, rinsed and allowed to recover (LLOME + recovery). Cells were incubated overnight, fixed the next day, and stained for actin (Alexa-488 phalloidin). Fluorescence was analyzed using a BioTek imaging microplate reader. y-axis represents Alexa-488 nm fluorescence [AU]. Data were analyzed with two-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance. *p < 0.05.

    Techniques Used: Transfection, Plasmid Preparation, Control, Incubation, Staining, Confocal Microscopy, Fluorescence, Imaging



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    ARF GTPases are recruited to damaged lysosomes. (A) HeLa cells were transfected with HA-tagged ARF1, ARF5, or ARF6 and treated the next day with 0.5 mM LLOME for 1 h or 3 h. At each time point, cells were fixed and stained for HA (cyan) and endogenous LAMP1 (magenta) and imaged with super-resolution confocal microscopy (Nikon NSPARC system). Scale bar indicates 10 µm for original size images and 1 µm for zoomed images. (B) Quantification of data in (A) n = 15 cells. Error bars represent mean +SD. Data were analyzed with one-way ANOVA. *p < 0.05 (C) (left) HEK293 cells stably expressing 3XHA-tagged TMEM192 were transfected with GFP-tagged ARF1, ARF5, or ARF6 constructs. Untreated and LLOME-treated (3 h) cells were subjected to lysosome immunoprecipitation using HA-conjugated magnetic beads (LysoIP). Total cell lysates and immunoprecipitates were immunoblotted for GFP to detect bound ARFs (left). Lysates and immunoprecipitates were also immunoblotted for endogenous LAMP1, Golgin-97, or EEA1 (right). (D) Quantification of data in (C) . Bars indicate mean +SD of ≥2 experimental replicates. Statistical analysis was performed using unpaired t-test. *p < 0.05. Statistics represent comparison of untreated HEK293 cells with 3-h LLOME (1 mM) treated cells quantified by densitometry. (E) HEK293 cells were treated with LLOME or not and subjected to the LysoIP protocol. Lysates were immunoblotted and stained for endogenous ARF5 and HA. (F) Quantification of data in (E) . Bars indicate mean +SD of three experimental replicates. *p < 0.05. Statistics represent comparison of untreated HEK293 cells with 20-min and 3-h LLOME-treated cells quantified by densitometry.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Loss of ARF5 impairs recovery after lysosomal damage

    doi: 10.3389/fmolb.2025.1699266

    Figure Lengend Snippet: ARF GTPases are recruited to damaged lysosomes. (A) HeLa cells were transfected with HA-tagged ARF1, ARF5, or ARF6 and treated the next day with 0.5 mM LLOME for 1 h or 3 h. At each time point, cells were fixed and stained for HA (cyan) and endogenous LAMP1 (magenta) and imaged with super-resolution confocal microscopy (Nikon NSPARC system). Scale bar indicates 10 µm for original size images and 1 µm for zoomed images. (B) Quantification of data in (A) n = 15 cells. Error bars represent mean +SD. Data were analyzed with one-way ANOVA. *p < 0.05 (C) (left) HEK293 cells stably expressing 3XHA-tagged TMEM192 were transfected with GFP-tagged ARF1, ARF5, or ARF6 constructs. Untreated and LLOME-treated (3 h) cells were subjected to lysosome immunoprecipitation using HA-conjugated magnetic beads (LysoIP). Total cell lysates and immunoprecipitates were immunoblotted for GFP to detect bound ARFs (left). Lysates and immunoprecipitates were also immunoblotted for endogenous LAMP1, Golgin-97, or EEA1 (right). (D) Quantification of data in (C) . Bars indicate mean +SD of ≥2 experimental replicates. Statistical analysis was performed using unpaired t-test. *p < 0.05. Statistics represent comparison of untreated HEK293 cells with 3-h LLOME (1 mM) treated cells quantified by densitometry. (E) HEK293 cells were treated with LLOME or not and subjected to the LysoIP protocol. Lysates were immunoblotted and stained for endogenous ARF5 and HA. (F) Quantification of data in (E) . Bars indicate mean +SD of three experimental replicates. *p < 0.05. Statistics represent comparison of untreated HEK293 cells with 20-min and 3-h LLOME-treated cells quantified by densitometry.

    Article Snippet: The antibodies we used were: rabbit antibody to LAMP1 (D2D11) (Cell Signaling Technology, Inc.), Cat No. 9091S, IF (1:400) WB (1:1200); mouse antibody to LAMP1 (1D4B) from Developmental Studies Hybridoma Bank; mouse antibody to GFP (Proteintech) Cat No.:66002-1-Ig WB (1:100,000); mouse antibody to HA (16B12) (Biolegend) WB (1:4000); rabbit antibody to mCherry (Sigma-Aldrich) Cat No.SAB2702295-100UL WB (1:10,000); rabbit antibody to OSBP (Sigma) Cat no. HPA039227 WB (1:1100) IF (1:100); rabbit antibody to ORP9 from Dr. Neale Ridgway, Dalhousie University, IF (1:1000); mouse antibody to Golgin97 (Molecular probes) Cat No. CDF4 A-21270 WB (1:500); rabbit antibody to ARF5 (Novus Biologicals) Cat No. NBP1-31005 WB (1:2500); sheep antibody to TGN46 (Serotec, Oxford United Kingdom); mouse antibody to Gal-3 (B-2) (Santa Cruz Biotechnology, Inc.) Cat No. sc-25279 IF (1:100); IRDye 800CW donkey anti-mouse secondary antibody (Li-COR) 926-32212 WB (1:10,000); IRDye 680RD goat anti-rabbit secondary antibody (Li-COR) 926-68071 WB (1:10,000); Alexa Fluor 488 donkey anti-mouse (ThermoFisher Scientific (Rockford, IL) IF (1:100); Alexa Fluor 488 donkey anti-rabbit (ThermoFisher Scientific (Rockford, IL) IF (1:100); Alexa Fluor 568 donkey anti-mouse (ThermoFisher Scientific (Rockford, IL) IF (1:100); Alexa Fluor 568 donkey anti-rabbit (ThermoFisher Scientific (Rockford, IL) IF (1:100).

    Techniques: Transfection, Staining, Confocal Microscopy, Stable Transfection, Expressing, Construct, Immunoprecipitation, Magnetic Beads, Comparison

    Lipid transfer proteins OSBP and ORP9 are recruited to damaged lysosomes in an ARF-independent manner. (A) HeLa cells were transfected with empty vector (shCTRL) or pLKO containing ARF1 or ARF5 shRNAs. Cells were treated or not with LLOME (0.5 mM) for 35 min, fixed and stained for endogenous OSBP (green), LAMP1 (red), and TGN46 (magenta), and imaged with confocal microscopy. Scale bars indicate 10 µm. (B) HEK293 cells stably expressing tagged TMEM192 were transfected with empty pLKO vector (shCTRL) or pLKO containing ARF1 or ARF5 shRNAs. Untreated and LLOME (1 mM)-treated cells were subjected to the LysoIP protocol. Precipitated lysosomes were immunoblotted for endogenous OSBP. (C) HeLa cells were transfected with empty vector (shCTRL), ARF5, or ARF6 shRNAs. Cells were treated or not with LLOME (0.5 mM) for 20 min, fixed and stained for ORP9 (green), LAMP1 (red), and F-actin (blue), and imaged with confocal microscopy. Scale bars indicate 10 µm. (D) Quantification of data in (C) n = 28 cells. Error bars represent mean +SD. Data were analyzed with two-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance. *p < 0.05.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Loss of ARF5 impairs recovery after lysosomal damage

    doi: 10.3389/fmolb.2025.1699266

    Figure Lengend Snippet: Lipid transfer proteins OSBP and ORP9 are recruited to damaged lysosomes in an ARF-independent manner. (A) HeLa cells were transfected with empty vector (shCTRL) or pLKO containing ARF1 or ARF5 shRNAs. Cells were treated or not with LLOME (0.5 mM) for 35 min, fixed and stained for endogenous OSBP (green), LAMP1 (red), and TGN46 (magenta), and imaged with confocal microscopy. Scale bars indicate 10 µm. (B) HEK293 cells stably expressing tagged TMEM192 were transfected with empty pLKO vector (shCTRL) or pLKO containing ARF1 or ARF5 shRNAs. Untreated and LLOME (1 mM)-treated cells were subjected to the LysoIP protocol. Precipitated lysosomes were immunoblotted for endogenous OSBP. (C) HeLa cells were transfected with empty vector (shCTRL), ARF5, or ARF6 shRNAs. Cells were treated or not with LLOME (0.5 mM) for 20 min, fixed and stained for ORP9 (green), LAMP1 (red), and F-actin (blue), and imaged with confocal microscopy. Scale bars indicate 10 µm. (D) Quantification of data in (C) n = 28 cells. Error bars represent mean +SD. Data were analyzed with two-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance. *p < 0.05.

    Article Snippet: The antibodies we used were: rabbit antibody to LAMP1 (D2D11) (Cell Signaling Technology, Inc.), Cat No. 9091S, IF (1:400) WB (1:1200); mouse antibody to LAMP1 (1D4B) from Developmental Studies Hybridoma Bank; mouse antibody to GFP (Proteintech) Cat No.:66002-1-Ig WB (1:100,000); mouse antibody to HA (16B12) (Biolegend) WB (1:4000); rabbit antibody to mCherry (Sigma-Aldrich) Cat No.SAB2702295-100UL WB (1:10,000); rabbit antibody to OSBP (Sigma) Cat no. HPA039227 WB (1:1100) IF (1:100); rabbit antibody to ORP9 from Dr. Neale Ridgway, Dalhousie University, IF (1:1000); mouse antibody to Golgin97 (Molecular probes) Cat No. CDF4 A-21270 WB (1:500); rabbit antibody to ARF5 (Novus Biologicals) Cat No. NBP1-31005 WB (1:2500); sheep antibody to TGN46 (Serotec, Oxford United Kingdom); mouse antibody to Gal-3 (B-2) (Santa Cruz Biotechnology, Inc.) Cat No. sc-25279 IF (1:100); IRDye 800CW donkey anti-mouse secondary antibody (Li-COR) 926-32212 WB (1:10,000); IRDye 680RD goat anti-rabbit secondary antibody (Li-COR) 926-68071 WB (1:10,000); Alexa Fluor 488 donkey anti-mouse (ThermoFisher Scientific (Rockford, IL) IF (1:100); Alexa Fluor 488 donkey anti-rabbit (ThermoFisher Scientific (Rockford, IL) IF (1:100); Alexa Fluor 568 donkey anti-mouse (ThermoFisher Scientific (Rockford, IL) IF (1:100); Alexa Fluor 568 donkey anti-rabbit (ThermoFisher Scientific (Rockford, IL) IF (1:100).

    Techniques: Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Stable Transfection, Expressing

    ARF5-depleted cells do not recover from lysosomal damage. (A) Schematic of Gal3 recovery assay. (B) HeLa cells were transfected with empty vector control (shCTRL), ARF5, or ARF6 shRNAs. Cells were treated or not with LLOME (0.5 mM) which was rinsed out after 20 min of incubation. Cells were allowed to recover for either 1 h or 8 h, after which they were fixed and stained for endogenous Gal3 (cyan), LAMP1 (magenta), and F-actin (yellow) and imaged with confocal microscopy. Scale bar indicates 10 µm. (C) Quantification of data in (B) n = 40–66 cells from two independent experiments. Error bars represent mean +SD. Data were analyzed with nonparametric Kruskal–Wallis one-way ANOVA test with Dunn’s correction. *p < 0.05 (D) HeLa cells were transfected with empty vector (shCTRL), ARF5, or ARF6 shRNAs and plated in black-walled 96-well plates. Cells were then treated with LLOME overnight or after 20 min of incubation, rinsed and allowed to recover (LLOME + recovery). Cells were incubated overnight, fixed the next day, and stained for actin (Alexa-488 phalloidin). Fluorescence was analyzed using a BioTek imaging microplate reader. y-axis represents Alexa-488 nm fluorescence [AU]. Data were analyzed with two-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance. *p < 0.05.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Loss of ARF5 impairs recovery after lysosomal damage

    doi: 10.3389/fmolb.2025.1699266

    Figure Lengend Snippet: ARF5-depleted cells do not recover from lysosomal damage. (A) Schematic of Gal3 recovery assay. (B) HeLa cells were transfected with empty vector control (shCTRL), ARF5, or ARF6 shRNAs. Cells were treated or not with LLOME (0.5 mM) which was rinsed out after 20 min of incubation. Cells were allowed to recover for either 1 h or 8 h, after which they were fixed and stained for endogenous Gal3 (cyan), LAMP1 (magenta), and F-actin (yellow) and imaged with confocal microscopy. Scale bar indicates 10 µm. (C) Quantification of data in (B) n = 40–66 cells from two independent experiments. Error bars represent mean +SD. Data were analyzed with nonparametric Kruskal–Wallis one-way ANOVA test with Dunn’s correction. *p < 0.05 (D) HeLa cells were transfected with empty vector (shCTRL), ARF5, or ARF6 shRNAs and plated in black-walled 96-well plates. Cells were then treated with LLOME overnight or after 20 min of incubation, rinsed and allowed to recover (LLOME + recovery). Cells were incubated overnight, fixed the next day, and stained for actin (Alexa-488 phalloidin). Fluorescence was analyzed using a BioTek imaging microplate reader. y-axis represents Alexa-488 nm fluorescence [AU]. Data were analyzed with two-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance. *p < 0.05.

    Article Snippet: The antibodies we used were: rabbit antibody to LAMP1 (D2D11) (Cell Signaling Technology, Inc.), Cat No. 9091S, IF (1:400) WB (1:1200); mouse antibody to LAMP1 (1D4B) from Developmental Studies Hybridoma Bank; mouse antibody to GFP (Proteintech) Cat No.:66002-1-Ig WB (1:100,000); mouse antibody to HA (16B12) (Biolegend) WB (1:4000); rabbit antibody to mCherry (Sigma-Aldrich) Cat No.SAB2702295-100UL WB (1:10,000); rabbit antibody to OSBP (Sigma) Cat no. HPA039227 WB (1:1100) IF (1:100); rabbit antibody to ORP9 from Dr. Neale Ridgway, Dalhousie University, IF (1:1000); mouse antibody to Golgin97 (Molecular probes) Cat No. CDF4 A-21270 WB (1:500); rabbit antibody to ARF5 (Novus Biologicals) Cat No. NBP1-31005 WB (1:2500); sheep antibody to TGN46 (Serotec, Oxford United Kingdom); mouse antibody to Gal-3 (B-2) (Santa Cruz Biotechnology, Inc.) Cat No. sc-25279 IF (1:100); IRDye 800CW donkey anti-mouse secondary antibody (Li-COR) 926-32212 WB (1:10,000); IRDye 680RD goat anti-rabbit secondary antibody (Li-COR) 926-68071 WB (1:10,000); Alexa Fluor 488 donkey anti-mouse (ThermoFisher Scientific (Rockford, IL) IF (1:100); Alexa Fluor 488 donkey anti-rabbit (ThermoFisher Scientific (Rockford, IL) IF (1:100); Alexa Fluor 568 donkey anti-mouse (ThermoFisher Scientific (Rockford, IL) IF (1:100); Alexa Fluor 568 donkey anti-rabbit (ThermoFisher Scientific (Rockford, IL) IF (1:100).

    Techniques: Transfection, Plasmid Preparation, Control, Incubation, Staining, Confocal Microscopy, Fluorescence, Imaging

    HeLa cells were treated with 2 mM levacetylleucine (NALL) for 18 hours, then processed for In-Cell Western analysis: endogenous LAMP1 was labelled with an anti-LAMP1 antibody and stained with CellTag700 (for normalization to cell number). Data are expressed as the mean (LAMP1/CellTag700) ± SEM of 12 replicates, and significance determined by a paired t-test, with significance denoted by *** p < 0.001 compared to vehicle control (Ctrl), i.e. 0 mM NALL.

    Journal: bioRxiv

    Article Title: Stereospecific rapid activation of Transcription Factor EB (TFEB) by Levacetylleucine (NALL)

    doi: 10.1101/2025.06.06.657607

    Figure Lengend Snippet: HeLa cells were treated with 2 mM levacetylleucine (NALL) for 18 hours, then processed for In-Cell Western analysis: endogenous LAMP1 was labelled with an anti-LAMP1 antibody and stained with CellTag700 (for normalization to cell number). Data are expressed as the mean (LAMP1/CellTag700) ± SEM of 12 replicates, and significance determined by a paired t-test, with significance denoted by *** p < 0.001 compared to vehicle control (Ctrl), i.e. 0 mM NALL.

    Article Snippet: Following block in Odyssey blocking buffer (LICORbio), cells were incubated with mouse anti-LAMP1 antibody (H4A3, DSHB at The University of Iowa) and was detected using IRDye 800CW goat anti-mouse IgG secondary antibody (LICORbio).

    Techniques: In-Cell ELISA, Staining, Control

    A. Reconstructed micrographs of LAMP1 staining (grayscale) in resting and macrophages that engulfed filamentous Legionella (red). Phagosome-associated LAMP1 was defined as LAMP1 that colocalized onto and to the periphery of Legionella . Dashed lines contours the cell. Free lysosomes were interpreted as LAMP1 puncta not proximal to phagosomes. Scale bar = 10 µm. B. Quantification of LAMP1-puncta per cell as described in A. C, H. Macrophages were given no particles (Mock phagocytosis, lanes 1-4) or allowed to engulf BSA-anti-BSA-opsonized magnetic beads (Magnetic beads, lanes 5-8) for 2 h, followed by 1 h incubation to elicit maturation. Phagosomes were then magnetically isolated, washed with PBS, and analyzed with Western Blot. LAMP1 (C) and VAMP3 (H) abundance were probed as a proxy for lysosome and endosome content, respectively. GAPDH was used as a loading control in whole cell lysates (WCL). Post-magnetic lysate (PML) is content remaining in lysate after magnetic separation; Wash fractions (W); Magnetic phagosome fraction is represented by M. D, I : Quantification of percent LAMP1 (D) and VAMP3 (I) remaining in lysate after magnetic separation. Percent remaining protein is defined as the percent ratio of GAPDH-normalized protein content in the PML fraction (lanes 2 and 6 for mock phagocytosis and magnetic beads, respectively) to GAPDH-normalized protein content in the WCL fraction (lanes 1 and 5 for mock phagocytosis and magnetic beads, respectively). E, J : Analysis in C and H, respectively, but normalized to mock phagocytosis to account for experimental variation in absolute values. Data represented as mean ± STD of N=4 independent experiments. Sample means were compared using two-tailed, paired Student’s t-test. *: p<0.05; **: p<0.01 (D, I) or one sample t and Wilcoxon test (E, J). F. Macrophages were transfected to express VAMP3-GFP (magenta) and then were given no particles (resting) or allowed to engulf IgG-opsonized beads for 2 h. Cytosol was demarcated with CFSE staining (green). Phagosome-associated VAMP3 was defined as VAMP3 that colocalized to the phagosome periphery as defined by black void in CFSE resulting from the bead contour. Free endosomes were interpreted as small, VAMP3 puncta not proximal to phagosomes. Scale bar = 10 µm. G. Quantification of VAMP3-puncta per cell as described in E. Data represented as mean ± SEM of N=3 independent experiments using 24-40 individual cells per condition per replicate. Data was analysed two-tailed, paired Student’s t-test.

    Journal: bioRxiv

    Article Title: Depletion of endomembrane reservoirs drives phagocytic appetite exhaustion in macrophages

    doi: 10.1101/2024.07.31.605905

    Figure Lengend Snippet: A. Reconstructed micrographs of LAMP1 staining (grayscale) in resting and macrophages that engulfed filamentous Legionella (red). Phagosome-associated LAMP1 was defined as LAMP1 that colocalized onto and to the periphery of Legionella . Dashed lines contours the cell. Free lysosomes were interpreted as LAMP1 puncta not proximal to phagosomes. Scale bar = 10 µm. B. Quantification of LAMP1-puncta per cell as described in A. C, H. Macrophages were given no particles (Mock phagocytosis, lanes 1-4) or allowed to engulf BSA-anti-BSA-opsonized magnetic beads (Magnetic beads, lanes 5-8) for 2 h, followed by 1 h incubation to elicit maturation. Phagosomes were then magnetically isolated, washed with PBS, and analyzed with Western Blot. LAMP1 (C) and VAMP3 (H) abundance were probed as a proxy for lysosome and endosome content, respectively. GAPDH was used as a loading control in whole cell lysates (WCL). Post-magnetic lysate (PML) is content remaining in lysate after magnetic separation; Wash fractions (W); Magnetic phagosome fraction is represented by M. D, I : Quantification of percent LAMP1 (D) and VAMP3 (I) remaining in lysate after magnetic separation. Percent remaining protein is defined as the percent ratio of GAPDH-normalized protein content in the PML fraction (lanes 2 and 6 for mock phagocytosis and magnetic beads, respectively) to GAPDH-normalized protein content in the WCL fraction (lanes 1 and 5 for mock phagocytosis and magnetic beads, respectively). E, J : Analysis in C and H, respectively, but normalized to mock phagocytosis to account for experimental variation in absolute values. Data represented as mean ± STD of N=4 independent experiments. Sample means were compared using two-tailed, paired Student’s t-test. *: p<0.05; **: p<0.01 (D, I) or one sample t and Wilcoxon test (E, J). F. Macrophages were transfected to express VAMP3-GFP (magenta) and then were given no particles (resting) or allowed to engulf IgG-opsonized beads for 2 h. Cytosol was demarcated with CFSE staining (green). Phagosome-associated VAMP3 was defined as VAMP3 that colocalized to the phagosome periphery as defined by black void in CFSE resulting from the bead contour. Free endosomes were interpreted as small, VAMP3 puncta not proximal to phagosomes. Scale bar = 10 µm. G. Quantification of VAMP3-puncta per cell as described in E. Data represented as mean ± SEM of N=3 independent experiments using 24-40 individual cells per condition per replicate. Data was analysed two-tailed, paired Student’s t-test.

    Article Snippet: Afterwards, cells were incubated with a 1:100 dilution of rat anti-mouse LAMP1 antibodies (clone 1D4B, Developmental Studies Hybridoma Bank) for 1 h at 37 °C, washed three times with PBS, followed by incubation for 1 h at room temperature with 1:1000 fluorescently tagged secondary antibodies, and subjected to another round of washes with PBS.

    Techniques: Staining, Magnetic Beads, Incubation, Isolation, Western Blot, Control, Two Tailed Test, Transfection