plasmid constructs encoding usp7  (Vigene Biosciences)

Journal: bioRxiv

Article Title: The actin-binding protein drebrin interacts with the respiratory syncytial virus matrix protein and promotes virus particle release

doi: 10.64898/2026.10.05.756730

Figure Lengend Snippet: (A) HEp-2 cells were “mock” infected (NI) or infected with RSV for 24 h, at MOI 1 and processed for IP using M antibodies. Input (1 %), unbound (1 %) and bound (30 %) fractions were loaded on SDS-PAGE and a Western blot was performed using antibodies indicated on the left. Representative of three independent experiments. (B) HEp-2-Drebrin-YFP and HEp-2-YFP cells were infected with RSV for 24 h, at MOI 1 and processed for IP using anti-GFP nanobodies. Input (0,5 %), unbound (0,5 %) and bound (30 %) fractions were loaded on SDS-PAGE and a Western blot was performed using antibodies indicated on the left. Drebrin-YFP and YFP corresponding bands were indicated by an arrow on the left. Representative of three independent experiments.

Article Snippet: YFP-tagged DBN (drebrin-YFP) construct has been provided from addgene (#40359).

Techniques: Infection, SDS Page, Western Blot

Journal: bioRxiv

Article Title: The actin-binding protein drebrin interacts with the respiratory syncytial virus matrix protein and promotes virus particle release

doi: 10.64898/2026.10.05.756730

Figure Lengend Snippet: Drebrin promotes viral particle release . (A) Schematic representation of the release assay. HEp-2 cells were transfected with control or specific siRNAs and seeded onto Transwell inserts for 48h (step 1). Cells were then infected with RSV-mCherry at an MOI of 1 (step 2). After washes to remove non-internalized virus, the infected “donor” cells were then placed in contact with target cells (step 3). The number of infected “donor” and “target” cells were quantified based on mCherry fluorescence using the Incucyte S3 system at 20h (step4) and 38h (step 5) post-infection respectively. RSV release efficiency was calculated as the ratio of infected target cells to infected donor cells and normalized to the control siRNA condition. (B) HEp-2 cells were transfected with si NT or drebrin-specific siRNAs (si drebrin) and release assay was performed as described in A. Date are mean ± SD of 6 independent experiments, each point represents mean of triplicates. Y axis represents viral release efficiency (ratio of infected target cells to infected donor cells) normalized to the control siRNA condition. (C) release assay was performed with HEp-2-YFP and HEp-2-Drebrin-YFP cells. Data are mean ± SD of 4 independent experiments done in triplicates. Y axis represents viral release efficiency (ratio of infected target cells to infected donor cells) normalized to the control siRNA condition. (B-C) A one-sample t-test was performed. * : P < 0.05; ** : P < 0.001.

Article Snippet: YFP-tagged DBN (drebrin-YFP) construct has been provided from addgene (#40359).

Techniques: Release Assay, Transfection, Control, Infection, Virus, Fluorescence

Journal: bioRxiv

Article Title: The actin-binding protein drebrin interacts with the respiratory syncytial virus matrix protein and promotes virus particle release

doi: 10.64898/2026.10.05.756730

Figure Lengend Snippet: (A) diagrams of drebrin mutants are shown. ADFH, actin-depolymerizing factor homology domain. ABD1, actin binding domain 1. ABD2, actin binding domain 2. IDD intrinsically disordered domain. Constructs are YFP-tagged at the C-terminus. Numbering is for human drebrin E. (B) HEp-2 cells lines were established with the indicated plasmids and cells were infected with RSV for 24h at MOI 1 and processed for IP using anti-GFP nanobodies. Input (0,5 %), unbound (0,5 %) and bound (30 %) fractions were loaded on SDS-PAGE and Western blotting using antibodies indicated on the left. Representative of three independent experiments. (C) release assay was performed as in with cells expressing indicated plasmids. Data are mean ± SD of 6 independent experiments done in triplicates. Y axis represents viral release efficiency (ratio of infected target cells to infected donor cells) normalized to the control siRNA condition. One-Way ANOVA with Dunnett’s multiple comparisons test and Geisser-Greenhouse correction was performed, ns : P > 0.05, ** : P < 0.01, **** : P < 0.0001.

Article Snippet: YFP-tagged DBN (drebrin-YFP) construct has been provided from addgene (#40359).

Techniques: Binding Assay, Construct, Infection, SDS Page, Western Blot, Release Assay, Expressing, Control

Journal: bioRxiv

Article Title: The actin-binding protein drebrin interacts with the respiratory syncytial virus matrix protein and promotes virus particle release

doi: 10.64898/2026.10.05.756730

Figure Lengend Snippet: (A) Drebrin-YFP HEp-2 cells were uninfected or infected with RSV at MOI 1 for 24 h. Proteins associated with drebrin-YFP were affinity-purified using GFP nanobody-coupled agarose beads, identified by mass spectrometry, and quantified based on spectral counts obtained for each sample. A log2 fold change was calculated by comparing RSV-infected and uninfected conditions. Only proteins with at least two spectral counts in at least two biological replicates in either condition were included in the analysis. Statistical significance was assessed using a beta-binomial test adapted for spectral count data. Results are presented as a volcano plot. The horizontal dashed gray line indicates the statistical significance threshold ( P < 0.05), whereas the vertical dashed gray lines indicate a twofold difference between infected and uninfected samples. Cellular proteins used in C are indicated in blue. Viral RSV proteins are indicated in black. Results from three independent biological replicates (B) Left: Heat map of RT-qPCR analysis comparing mRNA levels of 17 candidate proteins in RSV-infected and uninfected cells. Blue indicates > 3-fold upregulation and yellow < 3-fold. Right: Heat map of viral replication analysis comparing RSV-mCherry signal in RSV-infected cells treated with interactors-specific siRNA and si NT. Blue indicates replication >140%, brown replication < 60% and yellow 60% < replication >140%. Complete data are shown in Fig S8. (C) HEp-2 cells were depleted with the indicated siRNAs and viral release assay was performed and compared with control conditions. One-Way ANOVA with Dunnett’s multiple comparisons test and Geisser-Greenhouse correction was performed relative to si NT. *** : P <0.001, ns : P >0,05.

Article Snippet: YFP-tagged DBN (drebrin-YFP) construct has been provided from addgene (#40359).

Techniques: Infection, Affinity Purification, Mass Spectrometry, Analysis, Quantitative RT-PCR, Release Assay, Control, Significance Assay