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

Journal: Current biology : CB

Article Title: A septin double ring controls the spatiotemporal organization of the ESCRT machinery in cytokinetic abscission

doi: 10.1016/j.cub.2019.05.050

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-CHMP4B , Santa Cruz , Cat# sc-134946, RRID:AB_10608470.

Techniques: Virus, Recombinant, Purification, Proximity Ligation Assay, Labeling, shRNA, Software

Journal: Microbiology Spectrum

Article Title: Mutation of Phenylalanine 23 of Newcastle Disease Virus Matrix Protein Inhibits Virus Release by Disrupting the Interaction between the FPIV L-Domain and Charged Multivesicular Body Protein 4B

doi: 10.1128/spectrum.04116-22

Figure Lengend Snippet: Detection of interaction between ESCRT proteins and the FPIV L-domain. HA-tagged ALIX and Flag-tagged M protein (M-WT or M-F23A) (A) or Myc-tagged M protein (M-WT or M-F23A) and Flag-tagged ESCRT protein (TSG101, EAP20, EAP30, EAP45, CHMP1A, CHMP1B, CHMP2A, CHMP2B, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP5, CHMP6, CHMP7, or IST1) (B to F) were cotransfected into HEK293T cells. At 48 hpt, the cells were lysed and immunoprecipitated by using an anti-Flag M2 affinity gel. The assays were performed twice (A and B) or three times (C to F). The resulting immunoprecipitates together with cell lysates were analyzed via Western blotting. The affinity ratio between M protein (M-WT or M-F23A) and each ESCRT component was calculated by dividing the quantity of copurified HA/Myc-tagged protein by the quantity of purified Flag-tagged protein. The affinity ratio of M-WT or M-F23A was set to 1, and the relative affinity ratios of other proteins were obtained by comparison.

Article Snippet: Rabbit polyclonal antibody against CHMP4B (A7402) was purchased from Abclonal (Wuhan, China).

Techniques: Immunoprecipitation, Western Blot, Purification, Comparison

Journal: Microbiology Spectrum

Article Title: Mutation of Phenylalanine 23 of Newcastle Disease Virus Matrix Protein Inhibits Virus Release by Disrupting the Interaction between the FPIV L-Domain and Charged Multivesicular Body Protein 4B

doi: 10.1128/spectrum.04116-22

Figure Lengend Snippet: Effects of endogenous CHMP4B knockdown on rSG10 virion release from cells infected at an MOI of 1 or 0.01. (A) Cell viability upon transfection with each indicated siRNA was evaluated via a CCK-8 assay. The value of the si-NC-transfected sample was set to 1. The values of si-CHMP4B- and si-CHMP4C-transfected samples were obtained by comparison with the si-NC-transfected sample. The significance of each difference was calculated by one-way ANOVA. (B) Vero cells were transfected with the indicated siRNA and harvested at 24 hpt for RNA isolation; the resulting RNA was subjected to RT-qPCR to evaluate the percentage of remaining mRNA for CHMP4B or CHMP4C after knockdown. The significance of each difference was calculated with one-way ANOVA. (C) At 48 hpt with the siRNA described above, Vero cells were harvested, and the WCLs were analyzed by Western blotting conducted using specific antibodies against endogenous CHMP4B. (D to F) After transfection with the indicated siRNAs, Vero cells were infected with the indicated rNDV at an MOI of 1 (D) or 0.01 (E and F). At 24 hpi, the extracellular viruses were collected from the infected cells and their titers were determined by titration on corresponding cells. The significance of each difference was calculated with one-way ANOVA. (G to I) The extracellular viruses described for panels D to F were also pelleted through ultracentrifugation. Equal amounts of virions and WCLs were subjected to Western blotting and the budding efficiencies were calculated as described above. The significance of each difference was calculated with one-way ANOVA. The values ± SD from triplicate experiments are shown.

Article Snippet: Rabbit polyclonal antibody against CHMP4B (A7402) was purchased from Abclonal (Wuhan, China).

Techniques: Knockdown, Infection, Transfection, CCK-8 Assay, Comparison, Isolation, Quantitative RT-PCR, Western Blot, Titration

Journal: Microbiology Spectrum

Article Title: Mutation of Phenylalanine 23 of Newcastle Disease Virus Matrix Protein Inhibits Virus Release by Disrupting the Interaction between the FPIV L-Domain and Charged Multivesicular Body Protein 4B

doi: 10.1128/spectrum.04116-22

Figure Lengend Snippet: siRNA duplexes used in this study

Article Snippet: Rabbit polyclonal antibody against CHMP4B (A7402) was purchased from Abclonal (Wuhan, China).

Techniques: Sequencing, Knockdown, Negative Control

Journal: bioRxiv

Article Title: Engineered Nanotopographies Induce Transient Openings in the Nuclear Membrane

doi: 10.1101/2024.08.13.605467

Figure Lengend Snippet: A) Time-lapse imaging shows U2OS cells expressing NLS-GFP undergoing nuclear membrane breach and repair. Notably, after 90 minutes, cells display signs of breaching with subsequent repair mechanisms relocalizing NLS-GFP to the nucleus after 2.5 hrs. U2OS cells. B) The cytoplasmic and nuclear NLS-GFP signal ratio illustrates the decreasing ratio after the rupture incident suggesting the repair mechanism. C) Overall schematic and time lapse of images of U2OS cells expressing NLS-GFP and cGAS-mCherry showing membrane breach and repair. The cGAS-mCherry binds to the DNA exposed to the cytoplasm upon breaching. Cells showing breaching after 5 hrs by mislocalization of NLS to the cytoplasm and showing the cGAS signals. Additionally, cells repair after 7 hrs while the cGAS remains in the breach sites which is indicated by the profile analysis of cGAS and NLS over the yellow line. Representative images of U2OS cells transfected with NLS-GP and cGAS-mChery showing the site of the membrane breach on nanopillars induced by the curvature. D) Profile intensity of the NLS and cGAS shows increased cytoplasmic to nuclear NLS ratio follows by sudden increase in the cGAS signals which confirms the presence of cGAS and NLS upon membrane opening. Additionally, after 7 hrs Ku-80 relocalized to the nucleus while cGAS signal is still present. E) Immunofluorescence microscopy of U2OS cells, viewed both from the top and the side. The nuclei are stained with blue DAPI stain; Ku-80, which signals nuclear envelope breach in green; and CHMP4B ESCRT-III protein which is implicated in nuclear membrane repair in red. The highlighted yellow region within the images shows the area of nuclear envelope disruption, characterized by gaps in the DAPI and Ku-80 where CHMP4B is localized, suggesting active repair processes. Side-view images further emphasize CHMP4B’s presence at the sites of nuclear breach and repair on the nanopillars.

Article Snippet: Antibodies used for immunofluorescence were Rabbit anti-Ku-80 (1:200, Cell Signaling), Mouse anti-Lamin A/C (1:400, Biolegend), and Rabbit anti-CHMP4B (1:100, Novus Biologicals).

Techniques: Imaging, Expressing, Membrane, Transfection, Immunofluorescence, Microscopy, Staining, Disruption