ascc3  (Proteintech)

Journal: bioRxiv

Article Title: Viral Infections Drive Functional Remodeling of Ribosome-Associated Proteins

doi: 10.64898/2026.01.29.701737

Figure Lengend Snippet: (A) Scatter plot showing the relationship between changes in RNA-binding protein (RBP) recruitment to poly(A)-tailed mRNAs at 18 hpi with SINV (y-axis) and changes in their recruitment to ribosomes (x-axis). (B) Volcano plot showing changes in polysome-associated proteins at 12 hpi with SINV. RBPs previously identified as specifically binding to viral RNAs are colored burgundy. DEAD-box and DExH-box helicases, as well as ASCC2 and ASCC3, which also bind viral RNAs, are highlighted in green and turquoise, respectively. Viral proteins are not shown on the volcano plot.

Article Snippet: Cells were incubated with 1X PBS-Tween20-0.3% (PBS-T) supplemented with BSA 5% for 30□min followed by incubation with ASCC3 primary antibody (Protein-tech 85130-2-RR 1:100 dilution) in same buffer for 1□h.

Techniques: RNA Binding Assay, Binding Assay

Journal: bioRxiv

Article Title: Viral Infections Drive Functional Remodeling of Ribosome-Associated Proteins

doi: 10.64898/2026.01.29.701737

Figure Lengend Snippet: (A) Measurement of mCherry fluorescence serves as an indicator of SINV replication in cells that were transfected with the indicated sgRNA to edit genes encoding the different components of the ribosome-associated quality control-trigger (RQT) complex. The cells were infected at a low MOI for 24 hours. The bar plot shows the mean of three biological replicates, with each replicate consisting of three to four technical replicates. The error bars represent the standard error of the mean. Two-tailed paired t-tests were performed to compare the mCherry signal in individual knockout cells to the signal in control knockout cells. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. “ns” indicates not significant. (B) Immunoblots showing the efficiency with which each subunit of the RQT complex was knocked out. The β-actin immunoblot serves as a loading control. (C) Cells were either uninfected or infected with SINV at a high MOI for 12 hours. Before collection, the cells were left untreated or treated with 2 µM of anisomycin for 15 minutes, and lysed in RIPA buffer supplemented with 10 mM N-Ethylmaleimide. Cell lysates were used for immunoblotting with antibodies against the ribosomal proteins eS10, uS10, uS3, and uS5. The ubiquitin-modified ribosomal proteins are indicated on the blots as brown colors. (D) Cells were transfected with either a control sgRNA or one targeting PKR. Then, the cells were infected, treated with anisomycin, and lysed as described in panel C. An immunoblot against eS10 is shown, with the ubiquitinated eS10 indicated. (E) Schematic representation of the signaling pathways potentially activated in response to ribosome collisions, along with their corresponding collided ribosome sensors. (F) A bar plot showing the quantification of mCherry fluorescence in cells transfected with the indicated sgRNAs for gene editing and then infected with SINV at a low MOI for 24 hours. The bar plot represents the mean of three biological replicates, each consisting of three to four technical replicates. Error bars and statistical tests are as described in Panel A. (G) Immunoblot analysis of SINV capsid protein levels in cells transfected with ZNF598 - or GCN1 -targeting sgRNAs. Two different sgRNAs were tested for GCN1 gene editing. The cells were infected at a high MOI for 13 hours. The immunoblots below show the efficiency of ZNF598 or GCN1 gene knockout, with the β-actin immunoblots serving as loading controls. (H) Immunoblot analysis of ZAKα-mediated p38 phosphorylation in cells infected with SINV at a high MOI for 13 hours. UV-C treatment was used as a positive control for phosphorylated p38. (I) Schematic representation of the ubiquitination and de-ubiquitination of uS3 and uS5 by RNF10 and USP10, respectively, in response to ribosome stalling during translation initiation and elongation. (J) Bar plot showing mCherry fluorescence quantification in cells transfected with the indicated sgRNAs and subsequently infected with SINV at a low MOI for 24 hours. This bar plot represents the mean of two or three biological replicates, with each replicate consisting of three to four technical replicates. The error bars and statistical tests are as described in Panel A. (K) Immunoblot analysis of SINV capsid protein levels in cells transfected with either RNF10 - or USP10 -targeting sgRNAs. The cells were infected at a high MOI for 12 hours. Immunoblots against uS3 and uS5 validate the effectiveness of the RNF10 and USP10 knockouts by revealing decreased and increased levels of the uS3/uS5 ubiquitinated forms, respectively. The β-actin immunoblot serves as a loading control. (L) Immunoblot analysis of uS3 ubiquitination in cells transfected with sgRNAs targeting either ASCC3 or ZNF598 . The cells were infected at a high MOI for 12 hours.

Article Snippet: Cells were incubated with 1X PBS-Tween20-0.3% (PBS-T) supplemented with BSA 5% for 30□min followed by incubation with ASCC3 primary antibody (Protein-tech 85130-2-RR 1:100 dilution) in same buffer for 1□h.

Techniques: Fluorescence, Transfection, Control, Infection, Two Tailed Test, Knock-Out, Western Blot, Ubiquitin Proteomics, Modification, Protein-Protein interactions, Gene Knockout, Phospho-proteomics, Positive Control

Journal: bioRxiv

Article Title: Viral Infections Drive Functional Remodeling of Ribosome-Associated Proteins

doi: 10.64898/2026.01.29.701737

Figure Lengend Snippet: (A) Functional enrichment analysis of ASCC3 -knockout cells using the STRING database . (B) Volcano plot showing changes in protein abundance in ASCC3 -knockout cells compared to control knockout cells. Proteins containing a signal peptide are colored turquoise and ASCC3 is colored fuchsia. (C) Scatterplot showing the relationship between changes in protein abundance in ZNF598 -knockout cells (x-axis) and ASCC3 -knockout cells (y-axis). Proteins containing a signal peptide are colored turquoise. ZNF598, ASCC3, and ASCC2 are colored fuchsia. (D) Quantification of all SINV proteins in cells lacking ASCC3 by mass spectrometry, compared to control cells. The cells were infected at a high MOI for 13 hours. (E) Cell lysates from control- and ASCC3 -knockout cells were separated by polyacrylamide gel electrophoresis. The glycoproteins were stained with Pro-Q Emerald 488 dye, and the resulting green fluorescent signals were imaged (see upper panel). Total proteins were subsequently stained with Coomassie Blue (lower panel). The bar plot shows the quantification of glycoprotein signal intensity and represents the mean of three biological replicates. (F) Control and ASCC3 -knockout cells were subjected to subcellular fractionation of the cytosol and membranes. An immunoblot was performed to analyze the subcellular localization of ASCC3. The quality of the cell fractionation was verified by immunoblotting the ER proteins STT3A and SRPRA. The β-actin immunoblot serves as a loading control. (G) Cells that were either left uninfected or infected with SINV at a high MOI for 13 hours were subjected to subcellular fractionation of the cytosol and membranes. An immunoblot was performed to analyze the subcellular localization of ASCC3 in response to viral infection. An immunoblot against ubiquitinated uS3 was performed to evaluate the partitioning of stalled ribosomes between the cytosol and the ER. The quality of the cell fractionation was verified by immunoblotting the ER protein DDRGK1.

Article Snippet: Cells were incubated with 1X PBS-Tween20-0.3% (PBS-T) supplemented with BSA 5% for 30□min followed by incubation with ASCC3 primary antibody (Protein-tech 85130-2-RR 1:100 dilution) in same buffer for 1□h.

Techniques: Functional Assay, Knock-Out, Quantitative Proteomics, Control, Mass Spectrometry, Infection, Polyacrylamide Gel Electrophoresis, Staining, Fractionation, Western Blot, Cell Fractionation

Journal: bioRxiv

Article Title: Viral Infections Drive Functional Remodeling of Ribosome-Associated Proteins

doi: 10.64898/2026.01.29.701737

Figure Lengend Snippet: (A) Representative images showing the localization of ASCC3 and SINV subgenomic RNA by combined immunofluorescence and in situ hybridization in HEK293T cells. Control and ASCC3 -knockout cells were either mock-infected or infected with SINV for 12 hours. (B) Fluorescence intensities of ASCC3, SINV RNA, and nuclear signals measured in over 90 cells shown in panel (A). (C) Correlation between ASCC3 and SINV RNA fluorescence signals in infected control cells, with a calculated Pearson coefficient of R = 0.66.

Article Snippet: Cells were incubated with 1X PBS-Tween20-0.3% (PBS-T) supplemented with BSA 5% for 30□min followed by incubation with ASCC3 primary antibody (Protein-tech 85130-2-RR 1:100 dilution) in same buffer for 1□h.

Techniques: Immunofluorescence, In Situ Hybridization, Control, Knock-Out, Infection, Fluorescence