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. 2024 Nov 30;22:573. doi: 10.1186/s12964-024-01949-4

Fig. 2.

Fig. 2

NSP14 induces NF-κB activation through the linear ubiquitination pathway. a FLAG-tagged NSP14 or pCMV-3Tag-8 vector was transfected into HEK293 cells. Cell lysates were immunoprecipitated with the anti-FLAG antibody and blotted with anti-FLAG, anti-ubiquitin (Ub), and anti-α-tubulin antibodies. b Strategies targeting K63-linked ubiquitination and linear ubiquitination. c Wild-type HEK293 cells, two UBC13 knockout, and two HOIP knockout HEK293 cell lines were transfected with FLAG-tagged NSP14 for 24 h. Real-time PCR was performed to determine the relative IL-8 mRNA levels. d Vector or FLAG-tagged NSP14 (NSP14-FLAG) was transfected into HOIP wild-type and knockout HEK293 cells. After 48 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and blotted with anti-FLAG, anti-linear ubiquitin, anti-HOIP, and anti-α-tubulin antibodies. e Wild-type and OTULIN knockout HEK293 cells were transfected with FLAG-tagged NSP14 for 24 h. Real-time PCR was performed to determine the relative IL-8 mRNA levels. f Wild-type and OTULIN knockout HEK293 cells were transfected with vector or NSP14-FLAG. After 48 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and blotted with anti-FLAG, anti-linear ubiquitin, anti-OTULIN, and anti-α-tubulin antibodies. g NSP14-FLAG was transfected with vector or HA-tagged OTULIN (OTULIN-HA) into HEK293 cells. After 48 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and blotted with anti-FLAG, anti-linear ubiquitin, anti-HA, and anti-α-tubulin antibodies. h HEK293 cells were transfected with NSP14-FLAG and OTULIN-HA, together with the NF-κB reporter and pRL-SV40. After 48 h, cells were harvested, and the relative reporter activity was determined by calculating the ratio of firefly luciferase to Renilla luciferase. Lysates were blotted with anti-FLAG, anti-HA and anti-α-tubulin antibodies. The p-value was calculated by one-way ANOVA followed by Tukey's multiple comparisons test (h) and two-way ANOVA followed by Sidak's multiple comparisons test (c, e). i Proposed model illustrating NSP14 linear ubiquitination regulated by LUBAC and OTULIN