rabbit anti chmp4b  (Novus Biologicals)

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

Journal: The Journal of cell biology

Article Title: Vps60 initiates alternative ESCRT-III filaments.

doi: 10.1083/jcb.202206028

Figure Lengend Snippet: Figure 7. CHMP5 activity in mammalian cells. (A) Confocal images of immunofluorescence staining against CHMP4B of Hela-Kyoto cells expressing CHMP5- mNeonGreen under isosmotic or hypertonic conditions. (B and C) Confocal images of immunofluorescence staining against LAMP1 or CHMP4B of Hela-Kyoto cells expressing CHMP5-mNeonGreen with or without LLOMe treatment. Zoom-in of the deconvoluted image in C is shown in Fig. 8 A. (D) Live-cell imaging of Hela-Kyoto cells expressing CHMP5-mNeonGreen during osmotic shock. (E) Live-cell imaging of Hela-Kyoto cells expressing CHMP5-mNeonGreen upon LLOMe treatment. (F) Quantification of experiments described in D and E. Scale bar: 10 μm; D n = 3 ROI ≥400; E n = 3 ROI ≥2,500.

Article Snippet: Rabbit monoclonal antibody against LAMP1 (1/1,000 dilution for immunofluorescence) was from Cell Signaling (9091), and rabbit polyclonal antibody against CHMP4B (1/300) was from Proteintech (13683-1-AP).

Techniques: Activity Assay, Immunofluorescence, Staining, Expressing, Live Cell Imaging

Journal: The Journal of cell biology

Article Title: Vps60 initiates alternative ESCRT-III filaments.

doi: 10.1083/jcb.202206028

Figure Lengend Snippet: Figure 8. CHMP5 and CHMP4B polymers have distinct dynamics in cells. (A) Decon- voluted confocal images of immunofluores- cence staining against LAMP1 or CHMP4B (same as Fig. 7 C, higher magnification) of Hela- Kyoto cells expressing CHMP5-mNeonGreen upon LLOMe treatment (scale bar: 10 μm, 2 μm inlet). (B) Live-cell imaging of Hela-Kyoto cells expressing CHMP4B-GFP and CHMP5- mScarlet-I undergoing nuclear envelop refor- mation and cytokinetic abscission (scale bar: 10 μm). Arrowheads indicate the reforming nu- clear envelope and the cytokinetic bridge. (C) Cartoon of the proposed model for function of Vps60-based ESCRT-III filaments.

Article Snippet: Rabbit monoclonal antibody against LAMP1 (1/1,000 dilution for immunofluorescence) was from Cell Signaling (9091), and rabbit polyclonal antibody against CHMP4B (1/300) was from Proteintech (13683-1-AP).

Techniques: Staining, Expressing, Live Cell Imaging