Journal: Journal of Virology

Article Title: Interaction of coronavirus E protein with BRD2 plays important regulatory roles in viral replication and induction of pro-inflammatory response

doi: 10.1128/jvi.02201-25

Figure Lengend Snippet: BET family genes are differentially induced by CoV infection. ( A ) H1299, DF1, IPEC-J2, and Vero cells were infected with IBV, PEDV, HCoV-OC43, or HCoV-229E (MOI ~2) or mock-treated with UV-inactivated viruses. Total RNA was extracted at the indicated time points, and RT-qPCR was performed to quantify viral genomic RNA (CoV gRNA) and BET family mRNAs (BRD2, BRD3, BRD4, and BRDT). Data were normalized to GAPDH mRNA (0 hpi) using the ΔΔCt method (ns, non-significant; *, P <0.05; **, P < 0.01; ***, P < 0.001). N.D., non-determined. ( B ) Cells were infected or mock-treated as in panel A , harvested at the indicated times, and analyzed by Western blot with the specified antibodies. β-actin served as a loading control. Protein ladder sizes (kDa) are shown.

Article Snippet: Antibodies against BRD2 (#5848), NF-κB p65 (#8242), Phospho-NF-κB p65 (#3033), IκBα (#4814), Phospho-IκBα (#5209), IL-6 (#12912), FLAG-Tag (#8146), GFP-Tag (#2956), myc-Tag (#2276), H3 (#4499), α-Tubulin (#3873), and β-actin (#4967) were from Cell Signaling Technology.

Techniques: Infection, Quantitative RT-PCR, Western Blot, Control

Journal: Journal of Virology

Article Title: Interaction of coronavirus E protein with BRD2 plays important regulatory roles in viral replication and induction of pro-inflammatory response

doi: 10.1128/jvi.02201-25

Figure Lengend Snippet: Interaction of the C-terminal region of BRD2 protein with the C-terminal region of CoV E protein. ( A ) 293T cells were transfected with pXJ40-FLAG, pXJ40-IBV E-FLAG, pXJ40-IBV E-T16A-FLAG, pXJ40-IBV E-A26F-FLAG, or pXJ40-BRD2. After 24 h, lysates were immunoprecipitated (IP) with anti-FLAG beads and analyzed by Western blot (anti-FLAG/anti-BRD2). Protein ladder sizes (kDa) are shown. Sizes of protein ladders in kDa were indicated on the left. ( B ) Diagram showing the putative functional domains in the full-length and three truncated BRD2 constructs, BRD2-N1, BRD2-C1, and BRD2-C2. ( C ) 293T cells were transfected with pXJ40-FLAG, pXJ40-IBV E-FLAG, or myc-tagged BRD2 constructs (full-length, N1, C1, C2). Lysates were immunoprecipitated with anti-FLAG and probed with anti-FLAG/anti-myc. Sizes of protein ladders in kDa were indicated on the left. ( D ) Diagram showing the putative functional domains in the full-length and two truncated IBV E constructs, IBV E-N, IBV E-C. ( E ) 2293T cells expressing myc-BRD2-C1 with GFP-tagged IBV E (full-length, N-, or C-terminus) were immunoprecipitated with anti-myc beads and blotted with anti-myc/anti-GFP. Sizes of protein ladders in kDa were indicated on the left.

Article Snippet: Antibodies against BRD2 (#5848), NF-κB p65 (#8242), Phospho-NF-κB p65 (#3033), IκBα (#4814), Phospho-IκBα (#5209), IL-6 (#12912), FLAG-Tag (#8146), GFP-Tag (#2956), myc-Tag (#2276), H3 (#4499), α-Tubulin (#3873), and β-actin (#4967) were from Cell Signaling Technology.

Techniques: Transfection, Immunoprecipitation, Western Blot, Functional Assay, Construct, Expressing

Journal: Journal of Virology

Article Title: Interaction of coronavirus E protein with BRD2 plays important regulatory roles in viral replication and induction of pro-inflammatory response

doi: 10.1128/jvi.02201-25

Figure Lengend Snippet: Re-localization of BRD2 from the nucleus to the cytoplasm in CoV-infected cells. ( A ) Cells infected with IBV (MOI~2) or mock-treated (UV-inactivated virus) were fixed at 0, 8, and 12 hpi, permeabilized, and immunostained with rabbit anti-BRD2 and mouse anti-IBV N/dsRNA. IBV N/dsRNA (Alexa Fluor 488, green), BRD2 (Alexa Fluor 594, red), and nuclei (DAPI/Hoechst 33,342, blue) were visualized. ( B ) Vero cells were either infected with IBV at an MOI~2 or mock-treated with UV-inactivated IBV and harvested at the indicated times post-infection. Nuclear and cytosolic fractions were prepared and subjected to western blotting with the indicated antibodies. Sizes of protein ladders in kDa were indicated on the left. ( C ) The grayscale values of protein bands shown in panel B were measured using Image J. The relative protein expression level of BRD2 in the nuclear fraction of the mock group was set as 100%, and the levels in other groups were calculated as percentages relative to this value.

Article Snippet: Antibodies against BRD2 (#5848), NF-κB p65 (#8242), Phospho-NF-κB p65 (#3033), IκBα (#4814), Phospho-IκBα (#5209), IL-6 (#12912), FLAG-Tag (#8146), GFP-Tag (#2956), myc-Tag (#2276), H3 (#4499), α-Tubulin (#3873), and β-actin (#4967) were from Cell Signaling Technology.

Techniques: Infection, Virus, Western Blot, Expressing

Journal: Journal of Virology

Article Title: Interaction of coronavirus E protein with BRD2 plays important regulatory roles in viral replication and induction of pro-inflammatory response

doi: 10.1128/jvi.02201-25

Figure Lengend Snippet: The association between the host protein BRD2 and IBV virions. ( A ) IBV-infected Vero cell supernatants were purified by sucrose gradient 20%. The sucrose pellet was analyzed by Western blot with the indicated antibodies. β-actin served as a loading control; protein ladder sizes (kDa) are shown. ( B ) IBV-infected Vero cell supernatants were purified by sucrose gradient (20%–60%). Fractions (12 total) were analyzed by Western blot with the indicated antibodies. Nine of twelve fractions (lanes 2–10) were 10× concentrated and analyzed by Western blot (antibodies indicated). β-actin was the loading control; protein ladder sizes in kDa are marked. ( C ) IBV-infected Vero cell supernatants were purified by sucrose gradient 20%. The sucrose pellet was immunoprecipitated with anti-IBV S/N/M/E and probed with anti-IBV S/N/M/E or anti-BRD2. Sizes of protein ladders in kDa were indicated on the left.

Article Snippet: Antibodies against BRD2 (#5848), NF-κB p65 (#8242), Phospho-NF-κB p65 (#3033), IκBα (#4814), Phospho-IκBα (#5209), IL-6 (#12912), FLAG-Tag (#8146), GFP-Tag (#2956), myc-Tag (#2276), H3 (#4499), α-Tubulin (#3873), and β-actin (#4967) were from Cell Signaling Technology.

Techniques: Infection, Purification, Western Blot, Control, Immunoprecipitation

Journal: Journal of Virology

Article Title: Interaction of coronavirus E protein with BRD2 plays important regulatory roles in viral replication and induction of pro-inflammatory response

doi: 10.1128/jvi.02201-25

Figure Lengend Snippet: BRD2 protein regulates CoV replication and apoptosis. ( A ) Cell viability of H1299-shNC/BRD2 and Vero-shNC/BRD2 cells was assessed by CCK-8 assay (A450). Significance levels were presented by the p -value (ns, non-significant; *, P <0.05; **, P < 0.01; ***, P < 0.001). N.D., non-determined. ( B ) IBV titers (log TCID50/ml) in lysates/supernatants of infected H1299-shNC/BRD2 and Vero-shNC/BRD2 cells at the indicated time points. Significance as described for panel A . ( C ) Western blot analysis of IBV-infected (MOI ~2) or mock-treated H1299-shNC/BRD2 and Vero-shNC/BRD2 cells using the indicated antibodies. Beta-actin was included as the loading control. Sizes of protein ladders in kDa were indicated on the left. ( D ) IBV-infected or mock-infected H1299 cells pre-transfected with siEGFP/siBRD2 were analyzed by Western blot. Beta-actin was included as the loading control. Sizes of protein ladders in kDa were indicated on the left. ( E ) Western blot of IBV-infected or mock-infected H1299 cells transfected with pXJ40-FLAG/pXJ40-FLAG-BRD2. Beta-actin was included as the loading control. Sizes of protein ladders in kDa were indicated on the left.

Article Snippet: Antibodies against BRD2 (#5848), NF-κB p65 (#8242), Phospho-NF-κB p65 (#3033), IκBα (#4814), Phospho-IκBα (#5209), IL-6 (#12912), FLAG-Tag (#8146), GFP-Tag (#2956), myc-Tag (#2276), H3 (#4499), α-Tubulin (#3873), and β-actin (#4967) were from Cell Signaling Technology.

Techniques: CCK-8 Assay, Infection, Western Blot, Control, Transfection

Journal: Journal of Virology

Article Title: Interaction of coronavirus E protein with BRD2 plays important regulatory roles in viral replication and induction of pro-inflammatory response

doi: 10.1128/jvi.02201-25

Figure Lengend Snippet: BRD2 promotes the induction of pro-inflammatory response in CoV-infected cells. ( A ) IBV-infected (MOI ~2) or mock-treated HeLa, DF1, and Vero cells were analyzed by RT-qPCR for viral gRNA and cytokine mRNAs (IL-6, IL-8, TNF-α), normalized to 0 hpi GAPDH. Significance levels were presented by the P -value (ns, non-significant; *, P <0.05; **, P < 0.01; ***, P < 0.001). N.D., non-determined. ( B ) Western blot analysis of IBV-infected or mock-treated HeLa, DF1, and Vero cells at the indicated time points. Beta-actin was included as the loading control. Sizes of protein ladders in kDa were indicated on the left. ( C ) RT-qPCR analysis of IBV RNAs (gRNA, sgRNA), cytokines (IL-6, IL-8, TNF-α), and BET family mRNAs (BRD2/3/4, BRDT) in H1299/Vero-shNC/BRD2 cells, normalized to GAPDH in shNC at 0 hpi. Significance as described for panel A . ( D ) IBV-infected or mock-infected H1299 cells pre-transfected with siEGFP/siBRD2 were analyzed by RT-qPCR for viral gRNA, cytokines, and BET mRNAs, normalized to GAPDH in siEGFP mock at 16 hpi. Significance as described for panel A .

Article Snippet: Antibodies against BRD2 (#5848), NF-κB p65 (#8242), Phospho-NF-κB p65 (#3033), IκBα (#4814), Phospho-IκBα (#5209), IL-6 (#12912), FLAG-Tag (#8146), GFP-Tag (#2956), myc-Tag (#2276), H3 (#4499), α-Tubulin (#3873), and β-actin (#4967) were from Cell Signaling Technology.

Techniques: Infection, Quantitative RT-PCR, Western Blot, Control, Transfection

Journal: Journal of Virology

Article Title: Interaction of coronavirus E protein with BRD2 plays important regulatory roles in viral replication and induction of pro-inflammatory response

doi: 10.1128/jvi.02201-25

Figure Lengend Snippet: BRD2 protein promotes the activation of the NF-κB signaling pathway and the expression of pro-inflammatory factors. ( A ) H1299 cells transfected with pXJ40-FLAG or pXJ40-FLAG-BRD2 were infected with IBV or mock-treated, then analyzed by Western blot. Beta-actin was included as the loading control. Sizes of protein ladders in kDa were indicated on the left. The FLAG, IBV N, and β-actin loading control shown here are from the same experimental batch as the samples in and are reused to ensure a consistent baseline for cross-figure comparison. ( B ) As in panel A , but infected with HCoV-OC43. Beta-actin was included as the loading control. Sizes of protein ladders in kDa were indicated on the left. ( C ) H1299 and Vero cells transfected as described for panel A were infected with IBV, PEDV, HCoV-OC43, or HCoV-229E. RT-qPCR measured viral gRNA, cytokine mRNAs (IL-6/8, TNF-α), and BET family transcripts (BRD2/3/4, BRDT), normalized to FLAG-transfected mock 16 hpi GAPDH. Significance levels were presented by the P -value (ns, non-significant; *, P <0.05; **, P < 0.01; ***, P < 0.001). N.D., non-determined.

Article Snippet: Antibodies against BRD2 (#5848), NF-κB p65 (#8242), Phospho-NF-κB p65 (#3033), IκBα (#4814), Phospho-IκBα (#5209), IL-6 (#12912), FLAG-Tag (#8146), GFP-Tag (#2956), myc-Tag (#2276), H3 (#4499), α-Tubulin (#3873), and β-actin (#4967) were from Cell Signaling Technology.

Techniques: Activation Assay, Expressing, Transfection, Infection, Western Blot, Control, Comparison, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Interaction of coronavirus E protein with BRD2 plays important regulatory roles in viral replication and induction of pro-inflammatory response

doi: 10.1128/jvi.02201-25

Figure Lengend Snippet: Diagram illustrating the current working model. Working model of BRD2-IκBα-NF-κB axis activation of pro-inflammatory cytokines/chemokines and BRD2-E protein interaction during CoV infection. Arrowheads indicate activation; blunt ends indicate suppression. Dashed lines represent incompletely characterized processes.

Article Snippet: Antibodies against BRD2 (#5848), NF-κB p65 (#8242), Phospho-NF-κB p65 (#3033), IκBα (#4814), Phospho-IκBα (#5209), IL-6 (#12912), FLAG-Tag (#8146), GFP-Tag (#2956), myc-Tag (#2276), H3 (#4499), α-Tubulin (#3873), and β-actin (#4967) were from Cell Signaling Technology.

Techniques: Activation Assay, Infection