Journal: Retrovirology

Article Title: Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers

doi: 10.1186/1742-4690-4-54

Figure Lengend Snippet: Subcellular localization of Gag and the gRNA . Subcellular fractionations of 293T cells expressing wild-type HIV-1 (A, as in (21)) or NC(ΔZF1ZF2) (B) or ΔNC (C) were analyzed by OptiPrep gradient centrifugation. Cells were broken as described in materials and methods and the post-nuclear supernatant (PNS) was fractionated by Optiprep gradient. 20 μl of each fraction were loaded on SDS-PAGE, and Gag and Lamp2 were analyzed by immunoblotting using anti-Cap24 and anti-Lamp2 antibodies. Each fraction of the gradient was tested for the presence of the gRNA by RT-PCR as described in materials and methods. The expected 132 bp DNA fragment was detected on 1% agarose gel. In addition to the gradient analyses, the immunofluorescence (IF) detections are shown, representing the cells stained with an anti-CAp24 (in green) for Gag (A) or mutated Gag (B and C), and the FISH treatment of the 293T cells expressing HIV-1 (A) or the NC Gag mutants (B and C) for the gRNA using the Gag-oligo-Cy3 probe (in red).

Article Snippet: Viral proteins were separated on 10% SDS-PAGE and detected by immunoblotting with a mouse anti-CAp24 (P3D10G9B8, BioMérieux), and the cellular protein in the gradient was detected with the mouse anti-Lamp2 (Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Gradient Centrifugation, SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Immunofluorescence, Staining

Journal: Journal of Advanced Research

Article Title: The ubiquitin-specific protease 8 antagonizes melatonin-induced endocytic degradation of MT 1 receptor to promote lung adenocarcinoma growth

doi: 10.1016/j.jare.2022.01.015

Figure Lengend Snippet: Endocytosed MT 1 is sorted through the endo-lysosomal pathway. A and B HeLa cells transfected with the GFP-MT 1 construct were treated with melatonin (1 mM) for indicated times. The colocalization of GFP-tagged MT 1 with endogenous EEA1 (A) and LAMP2 (B) was inspected by immunofluorescence and confocal microscopy. Nucleus was stained with DAPI. Representative confocal sections are shown with magnified insets to illustrate colocalization. Graphs on the right show the quantification of fluorescence intensities for GFP-MT 1 (green) and EEA1 or LAMP2 (red). Scale bar = 10 μm. C HeLa cells transiently expressing GFP-MT 1 were pretreated with chloroquine (CQ) for 30 min prior to melatonin exposure for 0, 6, and 12 h. Samples were then analyzed by confocal microscopy. Micrographs show representative confocal sections, with magnified insets demonstrating the colocalization of GFP-MT 1 with LAMP2. Graphs on the right demonstrate quantification. Scale bar = 10 μm.

Article Snippet: Mouse anti-LAMP2 antibody (ab25631, 1:50) was obtained from Abcam (Cambridge, UK).

Techniques: Transfection, Construct, Immunofluorescence, Confocal Microscopy, Staining, Fluorescence, Expressing