Fig. 3: Binding of NSP12 with RIPK1 promotes the activation of RIPK1.

a RIPK1 KO 293T cells were co-transfected with Myc-RIPK1 and Flag-NSP12 expression vectors as indicated for 20 h. The cell lysates in 1% NP40 lysis buffer were analyzed by immunoprecipitation using anti-Flag M2-affinity gel and followed by western blotting using indicated antibodies. b Calu3 cells were infected with SARS-CoV-2 at MOI of 1 for 36 h. The cell lysates in 1% NP40 lysis buffer were analyzed by immunoprecipitation using anti-RIPK1 antibody and followed by western blotting using indicated antibodies. c HeLa cells were transfected with Strep tag II-NSP12 expression vector in the absence or presence of Nec-1s for 24 h. The cell lysates were analyzed by western blotting using indicated antibodies. The arrow points to the band of p-S166 RIPK1. d HeLa cells were transfected with vector control or expression vectors of NSP12 variants (323P or 323L). The cell lysates were analyzed by western blotting using p-S166 RIPK1 antibody for detection of activated RIPK1 kinase. Actin served as a loading control. The arrow points to the band of p-S166 RIPK1. e RIPK1 KO 293T cells were co-transfected with 0.1 μg of Myc-RIPK1 expression vector and different amounts of Flag-NSP12-323P and Flag-NSP12-323L expression vectors as indicated. The cells were lysed with 2× Laemmli sample buffer and analyzed by western blotting using indicated antibodies as shown on the left. Densitometric analysis for the levels of RIPK1 activation (p-S166 RIPK1)/actin is shown on the right. Fold changes were calculated compared to expression of Myc-RIPK1 alone.