Journal: Autophagy

Article Title: Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis

doi: 10.1080/15548627.2023.2169309

Figure Lengend Snippet: Reagents and resources used in this study.

Article Snippet: pmRFP C1-CHMP4B , Sino Biological , HG14022-ANR.

Techniques: Protease Inhibitor, Electron Microscopy, Silver Staining, Plasmid Preparation, Negative Control, Subcloning, Recombinant, Expressing, shRNA

Journal: The Journal of cell biology

Article Title: Analysis of native Ist1 dynamics reveals multiple pools of ESCRT-III on endosomes

doi: 10.1083/jcb.202407013

Figure Lengend Snippet: (A) Cells natively expressing Ist1-HaloTag were fixed and stained using antibodies directed against Snx15 following labeling with the JF650-HaloTag ligand. Representative images are shown (left) with quantification highlighting the distribution of distances between structures labeled with each marker (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). Bar, 5 μm; inset bar, 2 μm. (B) Representative images of natively expressed Ist1-HaloTag in control cells and cells lacking Snx15 following labeling with the JF650-HaloTag ligand (left). Quantification of the number of Ist1-HaloTag–positive structures per unit area under each condition is also shown (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). Bar, 5 μm. (C) Quantification of CHMP1B levels in control cells and cells depleted of CHMP1A, CHMP1B, or both CHMP1A and CHMP1B, based on immunoblot analysis ( n = 3). Error bars represent the mean ± SEM. ****P < 0.0001 and ***P < 0.001, as calculated using a one-way ANOVA and Tukey’s post hoc test. (D) Representative images of natively expressed Ist1-HaloTag in control cells treated with a scrambled siRNA (Mock) or siRNAs targeting CHMP1 isoforms following labeling with JF650-HaloTag ligand (left). Quantification of the number of Ist1-HaloTag–positive structures per unit area under the conditions shown is also provided (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). ****P < 0.0001, as calculated using a two-sided t test. Bar, 5 μm. (E) Cells natively expressing Ist1-HaloTag were fixed and stained using antibodies directed against CHMP4B following labeling with JF650-HaloTag ligand and treatment with either a scrambled siRNA (Mock) or siRNAs targeting CHMP1A and CHMP1B. Representative images are shown (left) with quantification highlighting CHMP4B fluorescence intensity under each condition (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). ****P < 0.0001, as calculated using a two-sided t test. Bar, 5 μm; inset bar, 2 μm.

Article Snippet: Immunofluorescence studies were conducted as described previously using the following antibodies (1 μg/ml final concentration each): CHMP1A (rabbit polyclonal; Proteintech 15761-AP), CHMP1B (rabbit polyclonal; Proteintech 14639-1-AP), CHMP4B (rabbit polyclonal; Proteintech 13683-1-AP), Snx15 (rabbit polyclonal; Proteintech 16049-1-AP), spastin (mouse monoclonal; Santa Cruz Biotechnology SC-81624), GAPDH (mouse monoclonal; Proteintech 60004-1-Ig), and Ist1 (rabbit polyclonal; a gift of Dr. Wesley Sundquist, University of Utah, Salt Lake City, UT, USA).

Techniques: Expressing, Staining, Labeling, Marker, Control, Western Blot, Fluorescence

Journal: The Journal of cell biology

Article Title: Analysis of native Ist1 dynamics reveals multiple pools of ESCRT-III on endosomes

doi: 10.1083/jcb.202407013

Figure Lengend Snippet: (A and B) Confocal microscopy was used to monitor the fluorescence recovery of Ist1-HaloTag labeled with the JF650-HaloTag ligand after photobleaching in the presence or absence of EGF ( n = 30 each; 3 biological replicates). Representative images of individual Ist1-HaloTag–labeled structures are shown under both conditions (A), as well as fluorescence recovery curves (B). Error bars represent the mean ± SEM. Bar, 1 μm. (C) Quantification of Ist1-HaloTag fluorescence recovery after labeling with the JF650-HaloTag ligand following photobleaching in the presence or absence of Snx15 and EGF ( n = 30 each; 3 biological replicates). Error bars represent the mean ± SEM.

Article Snippet: Immunofluorescence studies were conducted as described previously using the following antibodies (1 μg/ml final concentration each): CHMP1A (rabbit polyclonal; Proteintech 15761-AP), CHMP1B (rabbit polyclonal; Proteintech 14639-1-AP), CHMP4B (rabbit polyclonal; Proteintech 13683-1-AP), Snx15 (rabbit polyclonal; Proteintech 16049-1-AP), spastin (mouse monoclonal; Santa Cruz Biotechnology SC-81624), GAPDH (mouse monoclonal; Proteintech 60004-1-Ig), and Ist1 (rabbit polyclonal; a gift of Dr. Wesley Sundquist, University of Utah, Salt Lake City, UT, USA).

Techniques: Diffusion-based Assay, Confocal Microscopy, Fluorescence, Labeling

Journal: Cell Reports

Article Title: Calcium-dependent ESCRT recruitment and lysosome exocytosis maintain epithelial integrity during Candida albicans invasion

doi: 10.1016/j.celrep.2021.110187

Figure Lengend Snippet:

Article Snippet: Rabbit anti-CHMP4B , Novus Biologicals , Cat#NBP1-91782; RRID: AB_11020392.

Techniques: Isolation, Virus, Recombinant, Electron Microscopy, Staining, Western Blot, Control, Plasmid Preparation, Software

Journal: The Journal of cell biology

Article Title: Analysis of native Ist1 dynamics reveals multiple pools of ESCRT-III on endosomes

doi: 10.1083/jcb.202407013

Figure Lengend Snippet: (A) Cells natively expressing Ist1-HaloTag were fixed and stained using antibodies directed against Snx15 following labeling with the JF650-HaloTag ligand. Representative images are shown (left) with quantification highlighting the distribution of distances between structures labeled with each marker (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). Bar, 5 μm; inset bar, 2 μm. (B) Representative images of natively expressed Ist1-HaloTag in control cells and cells lacking Snx15 following labeling with the JF650-HaloTag ligand (left). Quantification of the number of Ist1-HaloTag–positive structures per unit area under each condition is also shown (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). Bar, 5 μm. (C) Quantification of CHMP1B levels in control cells and cells depleted of CHMP1A, CHMP1B, or both CHMP1A and CHMP1B, based on immunoblot analysis ( n = 3). Error bars represent the mean ± SEM. ****P < 0.0001 and ***P < 0.001, as calculated using a one-way ANOVA and Tukey’s post hoc test. (D) Representative images of natively expressed Ist1-HaloTag in control cells treated with a scrambled siRNA (Mock) or siRNAs targeting CHMP1 isoforms following labeling with JF650-HaloTag ligand (left). Quantification of the number of Ist1-HaloTag–positive structures per unit area under the conditions shown is also provided (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). ****P < 0.0001, as calculated using a two-sided t test. Bar, 5 μm. (E) Cells natively expressing Ist1-HaloTag were fixed and stained using antibodies directed against CHMP4B following labeling with JF650-HaloTag ligand and treatment with either a scrambled siRNA (Mock) or siRNAs targeting CHMP1A and CHMP1B. Representative images are shown (left) with quantification highlighting CHMP4B fluorescence intensity under each condition (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). ****P < 0.0001, as calculated using a two-sided t test. Bar, 5 μm; inset bar, 2 μm.

Article Snippet: Immunofluorescence studies were conducted as described previously using the following antibodies (1 μg/ml final concentration each): CHMP1A (rabbit polyclonal; Proteintech 15761-AP), CHMP1B (rabbit polyclonal; Proteintech 14639-1-AP), CHMP4B (rabbit polyclonal; Proteintech 13683-1-AP), Snx15 (rabbit polyclonal; Proteintech 16049-1-AP), spastin (mouse monoclonal; Santa Cruz Biotechnology SC-81624), GAPDH (mouse monoclonal; Proteintech 60004-1-Ig), and Ist1 (rabbit polyclonal; a gift of Dr. Wesley Sundquist, University of Utah, Salt Lake City, UT, USA).

Techniques: Expressing, Staining, Labeling, Marker, Control, Western Blot, Fluorescence

Journal: The Journal of Cell Biology

Article Title: The ESCRT protein Chmp4c regulates mitotic spindle checkpoint signaling

doi: 10.1083/jcb.201709005

Figure Lengend Snippet: Chmp4c-depletion reduces cold-stable microtubule polymers. (A–D) Cells transfected with negative siRNA (control), Chmp4c siRNA (siChmp4c), Chmp4a siRNA (siChmp4a), or Chmp4b siRNA (siChmp4b) were treated with ice-cold medium for the indicated times. (E) Centromere length in metaphase-like spindles. Insets show magnified centromeres, and values indicate the centromere length. Bars: (inset) 0.5 µm. (F) Quantification of centromere length from cells as in E or incubated with nocodazole (nocod) for 4 h. Control: n = 291 centromeres; 20 cells; siChmp4c: n = 697 centromeres, 26 cells; nocodazole: n = 200 centromeres, 20 cells from three independent experiments. (G) Cells expressing GFP, WT or S210A mutant Chmp4c:GFP resistant to degradation by siChmp4c-2, or GFP:Vps4-K173Q were treated with ice-cold medium for 15 min. Mean tubulin intensity is shown, and values in the controls were set to one. n = 90 cells (B, D, and G) from three independent experiments. Error bars show the SD. ***, P < 0.001 compared with the control. Statistically significant differences were determined by ANOVA and Student’s t test. Bars, 5 µm.

Article Snippet: Human Chmp4c located in the open reading frame (5′-GCUUGGGCUACCUAAACUA-3′), Chmp4c-2 located in the 3′-untranslated region (5′-GUAGAGGAGUCUUAUAUGA-3′), ZW10 (a pool of three different siRNAs: 5′-GCAUGGAGCUCACAAUACA-3′, 5′-CCAAGUAUUUGCACCUUUA-3′, and 5′-CAAGCCAGCUUGCAAGAAA-3′), Chmp4a (a pool of three different siRNAs: 5′-CAAACUGACGGGACAUUAU-3′, 5′-CUGAGUGGGUAUCCUGAUA-3′, and 5′-GAAGGAUCUUGCUACAGAA-3′), and Chmp4b (a pool of three different siRNAs: 5′-CAACAUGGACAUCGAUAAA-3′, 5′-CAGUCCCUCUACCAAAUGU-3′, and 5′-GCUGAAUCCUCAAUGGAAA-3′) siRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Incubation, Expressing, Mutagenesis

Journal: Neoplasia (New York, N.Y.)

Article Title: Nephronectin is Correlated with Poor Prognosis in Breast Cancer and Promotes Metastasis via its Integrin-Binding Motifs

doi: 10.1016/j.neo.2018.02.008

Figure Lengend Snippet: Uncropped images of immunoblots displayed in main figures. (A) Uncropped version of immunoblot displayed in main B . Membranes were incubated with anti-V5 (CST) and GAPDH (Abcam) antibodies prior to development. Inset shows cropped area shown in main figure. (B-E) Uncropped versions of immunoblots displayed in main A . The membranes were cut horizontally prior to incubation with antibodies for ALIX (CST), V5 (CST), GAPDH (Abcam), GM130 (BD Biosciences) and CHMP4B (Atlas Antibodies/Sigma).

Article Snippet: IF detection was performed using anti-CHMP4B (Atlas Antibodies/Sigma, Cat.: HPA051751, Lot No.: R67106, dilution 1:300, anti-V5 (CST, Cat.: 13202S, Lot No.: 2, dilution 1:150) and secondary antibodies; Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647, (Thermo Fisher Scientific, Cat.: A32728, Lot No.: RJ243424, dilution 1:1000) and Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Thermo Fisher Scientific, Cat.: A32731, Lot No.: RJ243417, dilution 1:1000).

Techniques: Western Blot, Incubation

Journal: Neoplasia (New York, N.Y.)

Article Title: Nephronectin is Correlated with Poor Prognosis in Breast Cancer and Promotes Metastasis via its Integrin-Binding Motifs

doi: 10.1016/j.neo.2018.02.008

Figure Lengend Snippet: NPNT is localized in extracellular vesicles. (A) Western blot of isolated microvesicles and exosomes from 66cl4-cells expressing EV, NPNT wild-type, NPNT RGE, or NPNT RGE-AIA detected with anti-V5 antibodies (CST). ALIX and CHMP4B were used as markers for microvesicles and exosomes, GM130 as a negative control and GAPDH for normalization control. Whole-cell lysates of 66cl4-NPNT cells were included as control. The images are cropped to display only relevant bands. Full-length blots are shown in . IF showing colocalization between the exosomal marker CHMP4B and NPNT detected with anti-CHMP4B and NPNT antibodies (Abnova) and visualized with Alexa Fluor 647 and Alexa Fluor 488 secondary antibodies, respectively, of (B) lung samples from in vivo lung colonization assay and (C) MMTV-PyMT mammary tumor samples. Images are representative of a series from five mice. Nuclei were stained with Hoechst. -ab reflects a control with no primary antibody. Scale bar; 5 μm NPNT is localized in extracellular vesicles. (A) Western blot of isolated microvesicles and exosomes from 66cl4-cells expressing EV, NPNT wild-type, NPNT RGE, or NPNT RGE-AIA detected with anti-V5 antibodies (CST). ALIX and CHMP4B were used as markers for microvesicles and exosomes, GM130 as a negative control and GAPDH for normalization control. Whole-cell lysates of 66cl4-NPNT cells were included as control. The images are cropped to display only relevant bands. Full-length blots are shown in Supplementary Figure S3. IF showing colocalization between the exosomal marker CHMP4B and NPNT detected with anti-CHMP4B and NPNT antibodies (Abnova) and visualized with Alexa Fluor 647 and Alexa Fluor 488 secondary antibodies, respectively, of (B) lung samples from in vivo lung colonization assay and (C) MMTV-PyMT mammary tumor samples. Images are representative of a series from five mice. Nuclei were stained with Hoechst. -ab reflects a control with no primary antibody. Scale bar; 5 μm

Article Snippet: IF detection was performed using anti-CHMP4B (Atlas Antibodies/Sigma, Cat.: HPA051751, Lot No.: R67106, dilution 1:300, anti-V5 (CST, Cat.: 13202S, Lot No.: 2, dilution 1:150) and secondary antibodies; Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647, (Thermo Fisher Scientific, Cat.: A32728, Lot No.: RJ243424, dilution 1:1000) and Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Thermo Fisher Scientific, Cat.: A32731, Lot No.: RJ243417, dilution 1:1000).

Techniques: Western Blot, Isolation, Expressing, Negative Control, Marker, In Vivo, Staining