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. 2016 Dec 21;6:39420. doi: 10.1038/srep39420

Figure 4. Mutation K77R or S104A in M-SAD is sufficient to induce its interaction with RelAp43 and should be coupled with mutations D100A and M110L to abolish the M-SAD activation of the NF-κB reporter vector.

Figure 4

(A) HeLa cells were used to co-express V5-tagged RelAp43 in combination with the FLAG-tagged CAT as a negative control, M-Tha as a positive control or the indicated FLAG-tagged single, double or quadruple mutants of M-SAD. After 24 h, cells were lysed, protein expression in cell lysates was controlled (Input: WB V5 and WB 3xFLAG) and co-IP experiments were performed using anti-FLAG M2 beads. Interaction of RelAp43 with the different M constructs or CAT was visualized by western blot (IP: 3xFLAG; WB: V5). The western blots depicted here are the results of a representative experiment (three repeats). (B) Modulation of NF-κB activation in 293T cells in the presence of single, double or quadruple mutants of M-SAD compared to M-Tha, M-SAD or CAT. The NF-κB pathway was exogenously activated using 10 ng/mL TNF-α during 5 h (black bars) or left untreated (grey bars). The M protein of vaccine strain SAD with the TNF treatment was arbitrary considered as a reference. Significant results (p < 0.05) in comparison to untreated (*) and TNF treated (**) CAT transfected cells were annotated.