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

Journal: ACS chemical biology

Article Title: PROTAC-mediated selective degradation of cytosolic soluble epoxide hydrolase enhances ER-stress reduction

doi: 10.1021/acschembio.3c00017

Figure Lengend Snippet: The immunoblot results of sEH levels in HepG2 cells treated for 24 h with 1a (250 nM) together with: (A) proteasome inhibitor MG132, or (B) lysosome inhibitor BafA1. Student’s t-test or Wilcoxon-Mann-Whitney test was performed depending on the normality. Asterisks represent the significant difference between MG132 in the presence and absence of 1a and/or BafA1 (*: P < 0.05, **: P < 0.01, ***: P < 0.001), the hashtags represent the significant difference between treatment vs 1a (#: P < 0.05, ##: P < 0.01). (C) Immunofluorescence results of the colocalization of sEH with LAMP2. Left panel: the representative images of immunofluorescence; Right panel: the quantification results of immunofluorescence. The area of colocalization was normalized to the number of cells in the corresponding images. Scale bar, 25 mm. Mean ± SEM are shown. The difference between DMSO, 1a’, and 1a were analyzed using one-way ANOVA Holm Sidak test. The difference between 1a, and 1a plus BafA1 was analyzed by Student’s t-test.

Article Snippet: The following primary antibodies were used in this experiment: mouse monoclonal anti-sEH (sc-166961, Santa Cruz Biotechnology, Dallas, TX), LAMP2 rabbit mAb (#49067, Cell signaling, Danvers, MA), β-tubulin antibody (#2146, Cell signaling, Danvers, MA), Catalase rabbit mAb (#12980, Cell signaling, Danvers, MA).

Techniques: Western Blot, MANN-WHITNEY, Immunofluorescence