Journal: bioRxiv

Article Title: Characterization of individual HIV-1 budding event using ultra-fast atomic force microscopy reveals a multiplexed role for VPS4

doi: 10.1101/2021.12.12.472262

Figure Lengend Snippet: Analysis of the kinetics of single HIV-1 budding events in VPS4 and VTA1 knockout HeLa cells (VPS4A.KO, VPS4B.KO, and VTA1.KO) compared with wildtype (WT) HeLa cells. (A) Verifying VPS4/VTA1 depletion. WT or KO HeLa cells were directly lysed (left and middle panels) or transfected with an appropriate siRNA (siVps4B for VPS4A; siVps4A for VPS4B) and then lysed (right panel). Next, equal amounts of the lysed cells were loaded on SDS-PAGE and subjected to western blot analysis using the antibodies specified beneath each panel. (B) Testing viral production. The supernatants were collected from WT and KO HeLa cells transfected or not with siRNA probes, as indicated, and infected with HIV-1. Normalized amounts of supernatant were loaded on SDS-PAGE and subjected to western blot analysis using antibodies for the viral capsid protein p24. (C) Representative AFM images of cells depleted of different VPS4-related proteins, as indicated, and infected with HIV-1 particles. Budding events are marked with white arrows. Nearly all budding events had a low maximal height ( i.e ., produced short, as opposed to tall, buds). The average maximal heights for the labeled events were 10 nm, 27 nm, and 13 nm, respectively. Scale bar is 1 μm. (D–E) The relative populations of budding events producing tall and short particles (D) and having slow and fast kinetics (E) shown as a percentage (y-axis) and as the number of events (given on each bar). Average time spent in each of the three kinetic phases, growth, stationary, and decay, with the number of slow (F) and total (G) budding events shown on each bar. Error bars indicate SEM.

Article Snippet: Membranes were stained with anti-VPS4 (1:750, Sigma, SAB4200025), anti-P24 (1:10000, 183-H12-5C hybridoma ), or anti-VTA1 (1:1500, Invitrogen, PA5-21831) for 16 h at 4 °C, followed by peroxidase goat anti-mouse IgG (1:500, Jackson, Cat 115-035-166) or peroxidase goat anti-rabbit IgG (1:500, Jackson, Cat 111-035-144) for 1 hour at room temperature.

Techniques: Knock-Out, Transfection, SDS Page, Western Blot, Infection, Produced, Labeling

Journal: Molecular & Cellular Proteomics : MCP

Article Title: FMRP Long-Range Transport and Degradation Are Mediated by Dynlrb1 in Sensory Neurons

doi: 10.1016/j.mcpro.2023.100653

Figure Lengend Snippet: Genetic depletion of Dynlrb1 impairs FMRP degradation. A , representative images of cell bodies of cultured DRG neurons transduced with shControl or shDynlrb1 constructs. Transduced neurons are labeled by EYFP expressed by the viral constructs (in green ). FMRP granules are visualized by staining with an anti-FMRP antibody ( grayscale) . Scale bars represent 5 μm and 1 μm, respectively. B , quantification of FMRP intensity ( B i) and the number of FMRP-positive granules ( B ii) in the experiment described in ( A ). Mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, n = 3, unpaired t test. C , Western blot analysis of FMRP protein levels in shControl or shDynlrb1 DRG neurons after incubation with proteasomal inhibitor (MG132), lysosomal inhibitors (leupeptin, pepstatin A, and E-64d) or vehicles for 6 h. β-actin immunostaining was used to normalize FMRP levels. Viral transduction efficiency was visualized by an anti-GFP antibody. D , quantification of the experiment described in ( C ). Mean ± SEM, n = 5, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns not significant, one-way ANOVA followed by Tukey’s HSD post hoc correction for multiple comparisons. DRG, dorsal root ganglia; EYFP, enhanced YFP; FMRP, fragile X messenger ribonucleoprotein 1; HSD, honestly significant difference test.

Article Snippet: Primary antibodies used in this study are anti-Tyr-α-tubulin (Synaptic Systems GmbH; catalog no.: 302117), anti-βIII-tubulin (Synaptic Systems GmbH; catalog no.: 302304), anti-FMRP (Invitrogen; catalog no.: PA534584 & OTI1C6 clone, catalog no.: TA504290), anti-dynein intermediate chain (Chemicon International, clone IC74.1; catalog no.: MAB1618), anti-flag (Sigma–Aldrich, clone M2; catalog no.: F3165), anti–vacuolar protein sorting–associated protein 29 (Vps29) (Abcam; catalog no.: ab236796), anti–vacuolar protein sorting–associated protein VTA1 homolog (Vta1) (Invitrogen; catalog no.: PA521831), anti-dynein heavy chain (Proteintech; catalog no.: 12345-1-AP), antipuromycin (Merck; catalog no.: MABE343), anti–microtubule-associated protein 1b (Map1b) (Invitrogen; catalog no.: PA582798), anti-β-actin (Sigma–Aldrich; catalog no.: A5441), anti-GFP (Roche; catalog no.: 11814460001), anti-G3BP stress granule assembly factor 1 (G3bp1) (Sigma–Aldrich; catalog no.: G6046), anti-lysosomal-associated membrane protein 1 (Lamp1) ([D2D11] XP, Cell Signaling Technology; catalog no.: 9091S), and anti–annexin A11 (Anxa11) (Proteintech; catalog no.: 68089-1-IG).

Techniques: Cell Culture, Transduction, Construct, Labeling, Staining, Western Blot, Incubation, Immunostaining

Journal: bioRxiv

Article Title: Characterization of individual HIV-1 budding event using ultra-fast atomic force microscopy reveals a multiplexed role for VPS4

doi: 10.1101/2021.12.12.472262

Figure Lengend Snippet: Analysis of the kinetics of single HIV-1 budding events in VPS4 and VTA1 knockout HeLa cells (VPS4A.KO, VPS4B.KO, and VTA1.KO) compared with wildtype (WT) HeLa cells. (A) Verifying VPS4/VTA1 depletion. WT or KO HeLa cells were directly lysed (left and middle panels) or transfected with an appropriate siRNA (siVps4B for VPS4A; siVps4A for VPS4B) and then lysed (right panel). Next, equal amounts of the lysed cells were loaded on SDS-PAGE and subjected to western blot analysis using the antibodies specified beneath each panel. (B) Testing viral production. The supernatants were collected from WT and KO HeLa cells transfected or not with siRNA probes, as indicated, and infected with HIV-1. Normalized amounts of supernatant were loaded on SDS-PAGE and subjected to western blot analysis using antibodies for the viral capsid protein p24. (C) Representative AFM images of cells depleted of different VPS4-related proteins, as indicated, and infected with HIV-1 particles. Budding events are marked with white arrows. Nearly all budding events had a low maximal height ( i.e ., produced short, as opposed to tall, buds). The average maximal heights for the labeled events were 10 nm, 27 nm, and 13 nm, respectively. Scale bar is 1 μm. (D–E) The relative populations of budding events producing tall and short particles (D) and having slow and fast kinetics (E) shown as a percentage (y-axis) and as the number of events (given on each bar). Average time spent in each of the three kinetic phases, growth, stationary, and decay, with the number of slow (F) and total (G) budding events shown on each bar. Error bars indicate SEM.

Article Snippet: Membranes were stained with anti-VPS4 (1:750, Sigma, SAB4200025), anti-P24 (1:10000, 183-H12-5C hybridoma ), or anti-VTA1 (1:1500, Invitrogen, PA5-21831) for 16 h at 4 °C, followed by peroxidase goat anti-mouse IgG (1:500, Jackson, Cat 115-035-166) or peroxidase goat anti-rabbit IgG (1:500, Jackson, Cat 111-035-144) for 1 hour at room temperature.

Techniques: Knock-Out, Transfection, SDS Page, Western Blot, Infection, Produced, Labeling