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. 2021 Feb 11;17(2):e1009336. doi: 10.1371/journal.ppat.1009336

Fig 4. SUMOylation at K612 does not affect the stability of IAV PB1.

Fig 4

(A, C, and E) HEK293T cells were transfected with plasmids encoding WSN-H1PB1 or WSN-H1PB1/K612R (A), VN/1180-H5PB1 or VN/1180-H5PB1/K612R (C), or AH/1-H7PB1 or AH/1-H7PB1/K612R (E). At 24 h post-transfection, the cells were treated with 100 μg/ml cycloheximide (CHX). The steady-state levels of PB1 protein after CHX treatment were then assessed at the indicated time points by western blotting with an anti-PB1 mAb. Western blots of whole cell lysates with an anti-GAPDH mAb are shown as loading controls. (B, D, and F) HEK293T cells were transfected with plasmids encoding Flag-tagged WSN-H1PB1 or WSN-H1PB1/K612R (B), VN/1180-H5PB1 or VN1180-H5PB1/K612R (D), or AH/1-H7PB1 or AH/1-H7PB1/K612R (F), together with SUMO1 and Ubc9. At 24 h post-transfection, the cells were treated with 100 μg/ml CHX for the time indicated. The cell lysates were then immunoprecipitated with anti-Flag M2 agarose beads, and both the precipitated proteins and the whole cell lysates were western blotted by using an anti-PB1 mAb. Arrow, SUMOylated PB1 protein. Data shown are representative of three independent experiments.