addgene ha  (Addgene inc)

Journal: Frontiers in Cell and Developmental Biology

Article Title: Rab10-associated tubulation as an early marker for biogenesis of the assembly compartment in cytomegalovirus-infected cells

doi: 10.3389/fcell.2024.1517236

Figure Lengend Snippet: Depletion of EHBP1, but not Rabin8, prevents perinuclear expansion of Rab10-PD in pre-AC. (A) NIH 3T3 cells were transfected with siRNA for EHBP1 or Rabin8. Control cells were either not transfected (Ø), or transfected with “scrambled” siRNA. After 48 h, cells were infected with Δm138-MCMV (MOI of 10) for 16 h, or left uninfected, and lysed for Western blot analysis. Expression of EHBP1 and Rabin8 was determined on the same membrane along with pIE1 as a control for infection and β-actin as a loading control. (B) Immunofluorescence detection of endogenous EHBP1 and Rabin8 (red) in Scr. siRNA- and EHBP- or Rabin8-siRNA-treated cells at 16 h post-infection. Staining for pIE1 (green) served as a control for infection and DAPI for visualization of nuclei. Shown are the confocal images through the focal plane. (C) Immunofluorescence detection of endogenous Rab10 (red) in untreated (NT), Scr. siRNA- and EHBP1 or Rabin8 siRNA-treated cells. Staining for pIE1 (green) served as a control for infection and DAPI for visualization of nuclei. Shown are the confocal images through the focal plane. Bars, 25 μm. (D) Percentage of MCMV-infected (pIE1-positive) cells with perinuclear accumulation of Rab10 in Scr. siRNA- and EHBP1 or Rabin8 siRNA-treated cells 16 h post-infection, shown as mean ± SD. The total number of cells analysed: Scr 1,659; EHBP1 1,578; Rabin8 1,578. Ctrl., control level. Statistical significance was determined by Student’s t-test (**p < 0.01).

Article Snippet: Small interfering (si)RNA sequences were acquired as follows: negative (control) siRNA (1022076) was from Qiagen (Hilden, Germany); siRNA for Rab3IP/Rabin8 (Sc-152666) and siRNA for EHBP1 (Sc-144602) were from Santa Cruz Biotechnology Inc. (Dallas, United States); and siRNA for Rab10 (L-040862-01–0005) was from Dharmacon Inc. (Laffayete, Colorado, United States).

Techniques: Transfection, Control, Infection, Western Blot, Expressing, Membrane, Immunofluorescence, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Rab10-associated tubulation as an early marker for biogenesis of the assembly compartment in cytomegalovirus-infected cells

doi: 10.3389/fcell.2024.1517236

Figure Lengend Snippet: Depletion of Rab10 and EHBP1 does not prevent the establishment of pre-AC and the establishment of tubular intermediates downstream in the EE-to-ERC pathway. (A) Cells were transfected with either Scr, Rab10 (30 nM) or EHBP1 (100 nM) siRNA and infected with Δm138-MCMV (MOI of 10) 48 h after transfection. After 16 h, cells were fixed, permeabilized and stained with either Abs against Rab10, EHBP1 or PLEKHB2 (red fluorescence) in combination with Abs against pIE1 to control infection (green) and DAPI (blue) to stain nuclei. The merged images are shown, and the complete image sets are shown in . (B) The percentage of infected cells (IE1-positive) with perinuclear accumulation (pn.) of PLEKHB2 was determined by epifluorescence microscopy and presented as mean ± SD of seven (Rab10) and four (EHBP1) independent experiments. The numbers in the bars indicate the total number of cells analyzed.

Article Snippet: Small interfering (si)RNA sequences were acquired as follows: negative (control) siRNA (1022076) was from Qiagen (Hilden, Germany); siRNA for Rab3IP/Rabin8 (Sc-152666) and siRNA for EHBP1 (Sc-144602) were from Santa Cruz Biotechnology Inc. (Dallas, United States); and siRNA for Rab10 (L-040862-01–0005) was from Dharmacon Inc. (Laffayete, Colorado, United States).

Techniques: Transfection, Infection, Staining, Fluorescence, Control, Epifluorescence Microscopy