Journal: Advances in Dermatology and Allergology/Postȩpy Dermatologii i Alergologii

Article Title: Modulation of dermal equivalent of hypothalamus-pituitary-adrenal axis in mastocytosis

doi: 10.5114/ada.2021.107933

Figure Lengend Snippet: List of antibodies used in the study

Article Snippet: Anti-MCIR (Rb) , Abcam , ab125031 , 1 : 100 , ImmPRESS™ HRPAnti-Rabbit IgG (Peroxidase) Polymer Detection Kit.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Genomic tagging of endogenous human ESCRT-I complex preserves ESCRT-mediated membrane-remodeling functions

doi: 10.1074/jbc.RA119.009372

Figure Lengend Snippet: HIV-1 release and replication are not perturbed in GFP-Tsg101 knock-in cell lines. A–C , GFP-Tsg101 knock-in does not alter HIV-1 particle release and infectivity. For assessment of HIV-1 release and infectivity, HeLa cells were transiently transfected with the HIV-1 molecular clone pNL4-3. A , virus release assay. Cell and virion lysates were analyzed by SDS-PAGE and Western blotting for HIV-1 proteins, followed by chemiluminescent analysis. Positions of the HIV-1 Env glycoprotein precursor gp160, the HIV-1 Gag precursor Pr55Gag, the Gag processing intermediate p41, and HIV-1 p24(CA) are shown. GAPDH is shown as a loading control. B , virus release efficiencies ( VRE ) were calculated as the amount of virion-associated p24(CA) as a fraction of the total (cell plus virion) Gag protein detected and normalized to the result for WT HeLa; n = 3; mean ± S.D. ( error bars ). C , infectivity assay. Virus produced by HeLa clones was normalized for RT activity and used to infect TZM-bl cells, and luciferase activity was measured 2 days postinfection. n = 3; mean ± S.D. D–F , GFP-Tsg101 knock-in does not alter the Tsg101 dependence of HIV-1 particle release and infectivity. To assess the contributions of Tsg101 and ALIX, HeLa cells were transiently transfected with the WT HIV-1 molecular clone pNL4-3 (WT p6) or the L41A, L41R, or PTAP− p6 mutants. D , virus release assay, performed as in A. E , virus release efficiencies were calculated as in B and normalized to the result for WT p6 for each cell line; n = 3; mean ± S.D. F , infectivity assay, performed as in C ; n = 3; mean ± S.D. For the KI HeLa lines as for WT HeLa, PTAP− severely reduced the infectivity of the released virus, whereas L41A and L41R gave lesser defects in infectivity, showing that the dependence of HIV-1 infectivity on Tsg101 was not altered by the knock-in. G , GFP-Tsg101 knock-in does not disrupt HIV-1 replication in T cells. Spreading infection assays were performed in parental or knock-in Jurkat T cells transfected with WT or p6-mutant pNL4-3 molecular clones. Cultures were split, and supernatants were collected for RT analysis every 2–3 days.

Article Snippet: For the Tsg101 and ALIX Western blotting, the membranes were blocked with 5% milk in TBS, pH 7.4, and then probed with the mouse anti-Tsg101 or anti-ALIX primary antibodies (listed above) at 1:200 dilution in TBS, pH 7.4, with 5% milk and 0.05% Tween 20, followed by the goat anti-mouse HRP secondary antibody at 1:8000 dilution, and the secondary HRP conjugates were detected using ProSignal Femto ECL Reagent (Genesee Scientific (San Diego, CA), catalog no. 20-302).

Techniques: Knock-In, Infection, Transfection, Virus, Release Assay, SDS Page, Western Blot, Control, Produced, Clone Assay, Activity Assay, Luciferase, Mutagenesis