anti cpsf30  (Proteintech)

Journal: bioRxiv

Article Title: Host mRNA 3’-end processing machinery are critical binding partners during dengue virus infection

doi: 10.1101/2025.06.20.659860

Figure Lengend Snippet: a . Bar graph showing the enriched pathways associated with the human proteins identified in the DENV NS3 samples. Metascape analysis was performed as in . b . Protein-protein interaction network showing the interaction between NS3 and human protein complexes involved in mRNA cleavage, polyadenylation and splicing processes. The presence of an edge denotes the detection of the human protein in the NS3 samples, and the thickness of the edge represents the average CRAPome score of the interaction from the replicate samples for NS3. c . Heat map depicting the average CRAPome scores of the CPSF and U5 spliceosome complex members detected in the DENV NS3 and NS5 samples. d . V5 immunoprecipitation of 293T cells transfected with the indicated FLAG-tagged DENV proteins and V5-tagged proteins involved in RNA metabolism, followed by Western blotting with the indicated antibodies showing the specific interaction between CPSF3, CPSF4 and EFTUD2, and DENV NS3 and NS5.

Article Snippet: Cells were washed with 1x PBS before incubation with the following primary antibodies: dsRNA (1001050; Exalpha), DENV NS3 (3F8) , CPSF3 (H00051692-M01; Novus Biologicals), CPSF4 (15023-1-AP; Proteintech), RPN1 (198598; Abcam) and MAGT1 (17430-1-AP; Proteintech).

Techniques: Immunoprecipitation, Transfection, Western Blot

Journal: bioRxiv

Article Title: Host mRNA 3’-end processing machinery are critical binding partners during dengue virus infection

doi: 10.1101/2025.06.20.659860

Figure Lengend Snippet: a. Immunofluorescence assay of Huh7 cells infected with DENV showing intracellular distribution of CPSF3 or CPSF4 (green) with infected cells indicated by staining with NS3 (red). b. Quantification of the fluorescent intensity for CPSF proteins (green), DENV NS3 (red) and DAPI (blue) along the indicated line-scans was performed using the Zeiss ZEN software Profile function and plotted on a line graph using Prism. c. Immunofluorescence assay of Huh7 cells expressing DENV NS3 showing the intracellular distribution of CPSF3 or CPSF4 (green) with cells expressing NS3 indicated by straining with NS3’s FLAG tag (red). d. Quantification of the fluorescent intensity for CPSF proteins (green), FLAG (red) and DAPI (blue) along the indicated line-scans was performed using the Zeiss ZEN software Profile function and plotted on a line graph using Prism. For b and d, thirty individual data points of the fluorescent intensity for the CPSF proteins were taken from the middle of the nucleus and cytoplasm, and compared by multiple unpaired t-tests and a p-value less than 0.05 was considered significant (*, p < 0.05; ***, p < 0.001; ****, p < 0.0001). The images are representative of similar results from three independent biological experiments.

Article Snippet: Cells were washed with 1x PBS before incubation with the following primary antibodies: dsRNA (1001050; Exalpha), DENV NS3 (3F8) , CPSF3 (H00051692-M01; Novus Biologicals), CPSF4 (15023-1-AP; Proteintech), RPN1 (198598; Abcam) and MAGT1 (17430-1-AP; Proteintech).

Techniques: Immunofluorescence, Infection, Staining, Software, Expressing, FLAG-tag

Journal: bioRxiv

Article Title: Host mRNA 3’-end processing machinery are critical binding partners during dengue virus infection

doi: 10.1101/2025.06.20.659860

Figure Lengend Snippet: a . Immunofluorescence assay of DENV-infected Huh7 cells showing the intracellular distribution of CPSF4 (green) with infected cells indicated by staining with dsRNA (red). Quantification of the fluorescent intensity for CPSF proteins (green), dsRNA (red) and DAPI (blue) along the indicated line-scans was performed using the Zeiss ZEN software Profile function and plotted on a line graph using Prism (right). b. Immunofluorescence assay of Huh7 cells expressing DENV NS1 showing the levels of CPSF4 (green) in the nuclei of cells stained for the FLAG tag on NS1 (red). Quantification of the fluorescent intensity for CPSF proteins (green), FLAG (red) and DAPI (blue) along the indicated line-scans was performed using the Zeiss ZEN software Profile function and plotted on a line graph using Prism (right). Thirty individual data points of the fluorescent intensity for the CPSF proteins were taken from the middle of the nucleus and cytoplasm, and compared by multiple unpaired t-tests and a p-value less than 0.05 was considered significant (****, p < 0.0001). The images are representative of similar results from three independent biological experiments.

Article Snippet: Cells were washed with 1x PBS before incubation with the following primary antibodies: dsRNA (1001050; Exalpha), DENV NS3 (3F8) , CPSF3 (H00051692-M01; Novus Biologicals), CPSF4 (15023-1-AP; Proteintech), RPN1 (198598; Abcam) and MAGT1 (17430-1-AP; Proteintech).

Techniques: Immunofluorescence, Infection, Staining, Software, Expressing, FLAG-tag

Journal: bioRxiv

Article Title: Host mRNA 3’-end processing machinery are critical binding partners during dengue virus infection

doi: 10.1101/2025.06.20.659860

Figure Lengend Snippet: a . Bar graph (left) and dot plot (right) showing the relative levels of DENV genomic RNA and DENV plaque forming units (PFU) in Huh7 cells treated with the indicated siRNAs at 40μM for 24h before being infected with DENV for 24h. b. Bar graph (left) and dot plot (center) showing the relative levels of DENV genomic RNA and DENV plaque forming units (PFU) in Huh7 cells treated with increasing concentrations of siCPSF3-10. Bar graph showing the relative levels of CPSF3 mRNA and Western blot with the indicated antibodies showing the protein levels of DENV NS5 and CPSF3 (right). c . Bar graph (left) and dot plot (right) showing the relative levels of ZIKV genomic RNA and ZIKV plaque forming units (PFU) in Huh7 cells treated with the indicated siRNAs at 40μM for 24h before being infected with ZIKV for 24h. d . V5 immunoprecipitation of 293T cells transfected with FLAG-tagged ZIKV or DENV NS3 and V5-tagged CPSF3, followed by Western blotting with the indicated antibodies, showing that ZIKV NS3, like DENV NS3, interacts with CPSF3. e. Bar graph showing the relative cell viability of Huh7 cells treated with the indicated concentrations of JTE-607. f . Line graph showing the relative DENV infectivity of Huh7 cells treated with the indicated concentrations of JTE-607. The number of plaque forming units were counted for each sample and plotted relative to the untreated sample. The estimated EC 50 was calculated using Prism. Western blot with the indicated antibodies of the corresponding cell lysate samples showing the decrease in protein levels of DENV NS5, CPSF3, CPSF4 and actin with treatment of increasing concentrations of JTE-607. Mean values were compared to the respective control samples by Dunnett multiple comparison after one-way ANOVA, and a p-value less than 0.05 was considered significant (*, p < 0.05; **, p < 0.01; ****, p < 0.0001).

Article Snippet: Cells were washed with 1x PBS before incubation with the following primary antibodies: dsRNA (1001050; Exalpha), DENV NS3 (3F8) , CPSF3 (H00051692-M01; Novus Biologicals), CPSF4 (15023-1-AP; Proteintech), RPN1 (198598; Abcam) and MAGT1 (17430-1-AP; Proteintech).

Techniques: Infection, Western Blot, Immunoprecipitation, Transfection, Control, Comparison