anti lamp1 xp d2d11  (Cell Signaling Technology Inc)

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

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

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