Fig 10.

JINR1 interacts with the p65 subunit of NF-κB, JINR1 and RBM10 reciprocally regulate each other’s expression through NF-κB. (A) Upper panel: cell lysates from SH-SY5Y cells were immunoprecipitated with anti-RBM10 or IgG antibody. The immune complexes and the input were analyzed by immunoblotting with antibodies specific to p65, and the same membrane was stripped and reprobed to detect RBM10. Lower panel: protein prepared from SH-SY5Y cells was also immunoprecipitated with anti-p65 or IgG antibodies. The immune complexes and the input were analyzed by immunoblotting with an antibody specific to RBM10; the same membrane was stripped and reprobed to detect p65. A representative blot is shown from two independent experiments with similar results. (B) JINR1 interacts with the p65 subunit of NF-κB. JINR1 RNA levels in p65 immunoprecipitated from the lysates of formaldehyde-crosslinked MI or JEV-infected SH-SY5Y cells were measured by qRT-PCR analysis, normalized to input, and represented as fold enrichment relative to MI-IgG IP. Values represent mean ± SD from three independent experiments. *Significant change compared to MI-IgG IP (P < 0.05). #Significant change compared to MI-p65 IP (P < 0.05). (C) RBM10 depletion attenuates JEV-induced JINR1. qRT-PCR analysis of JINR1 upon RBM10 depletion in SH-SY5Y cells at 36 hpi. (D) RBM10 depletion attenuates DENV-induced JINR1. qRT-PCR analysis of JINR1 upon RBM10 depletion in SH-SY5Y cells at 36 hpi. (E) RBM10 depletion attenuates WNV-induced JINR1. qRT-PCR analysis of JINR1 upon RBM10 depletion in SH-SY5Y cells at 36 hpi. (F) RBM10 promotes the p65 recruitment at the promoter of JINR1 during JEV infection. Relative enrichment of p65 at the JINR1 promoter in MI or JEV-infected SH-SY5Y cells transfected with si-NS or si-RBM10 determined by ChIP-qRT-PCR at 36 hpi. Enrichment values are relative to MI-si-NS-IgG. (G) RBM10 promotes the p65 recruitment at the promoter of JINR1 during DENV infection. Relative enrichment of p65 at the JINR1 promoter in MI or DENV-infected SH-SY5Y cells transfected with si-NS or si-RBM10 determined by ChIP-qRT-PCR at 36 hpi. Enrichment values are relative to MI-si-NS-IgG. (H) RBM10 promotes the p65 recruitment at the promoter of JINR1 during WNV infection. Relative enrichment of p65 at the JINR1 promoter in MI or WNV-infected SH-SY5Y cells transfected with si-NS or si-RBM10 determined by ChIP-qRT-PCR at 36 hpi. Enrichment values are relative to MI-si-NS-IgG. (I) JINR1 promotes the p65 recruitment at the promoter of RBM10 during JEV infection. Relative enrichment of p65 at the RBM10 promoter in MI or JEV-infected SH-SY5Y cells transfected with ASO-NS or ASO-JINR1 determined by ChIP-qRT-PCR at 36 hpi. Enrichment values are relative to MI-ASO-NS-IgG. (J) JINR1 promotes the p65 recruitment at the promoter of RBM10 during DENV infection. Relative enrichment of p65 at the RBM10 promoter in MI or DENV-infected SH-SY5Y cells transfected with ASO-NS or ASO-JINR1 determined by ChIP-qRT-PCR at 36 hpi. Enrichment values are relative to MI-ASO-NS-IgG. (K) JINR1 promotes the p65 recruitment at the promoter of RBM10 during WNV infection. Relative enrichment of p65 at the RBM10 promoter in MI or WNV-infected SH-SY5Y cells transfected with ASO-NS or ASO-JINR1 determined by ChIP-qRT-PCR at 36 hpi. Enrichment values are relative to MI-ASO-NS-IgG. Error bars represent the mean ± SEM from three independent experiments. Statistical comparison was made using the Student’s t-test. (C and F) *Significant change compared to MI-si-NS, and #significant change compared to JEV-si-NS. (D and G) *Significant change compared to MI-si-NS, and #significant change compared to DENV-si-NS. (E and H) *Significant change compared to MI-si-NS, and #significant change compared to WNV-si-NS. (I) *Significant change compared to MI-ASO-NS, and #significant change compared to JEV-ASO-NS. (J) *Significant change compared to MI-ASO-NS, and #significant change compared to DENV-ASO-NS. (K) *Significant change compared to MI-ASO-NS, and #significant change compared to WNV-ASO-NS.