Figure 3
NiV V stabilizes UBXN1. (A) HEK293T cells were transfected with 400 ng of a vector expressing HA-tagged UBXN1 together with various amounts (100 ng, 200 ng, or 400 ng) of vector expressing NiV V or 400 ng NiV P. The total amount of transfected vector was kept constant by the addition of empty vector. After 24 h, the cells were lysed and the proteins were detected with western blotting. (B) HEK293T cells were transfected with 400 ng of a vector expressing for EGFP together with various amounts of vector expressing NiV V as described in (A). The proteins were detected with western blotting. (C) UBXN1 genes in HEK293 and 293 T cells were knocked out by the CRISPR-Cas9 system. The depletion of UBXN1 in 293UBXN1− and 293TUBXN1− cells was verified with western blotting. (D) 293TUBXN1−, Huh-7 and HeLa cells were transfected with 400 ng of a vector expressing HA-tagged UBXN1 together with various amounts (200 ng or 400 ng) of vector expressing NiV V. The total amount of transfected vector was kept constant by the addition of empty vector. After 24 h, the cells were lysed and the proteins were detected with western blotting. (E) HEK293T cells were transfected with 400 ng of a vector expressing for HA-tagged UBXN1 together with 400 ng of a vector expressing NiV V or an empty vector. Then the cells were treated with CHX for 0, 1, 2, 3, or 4 h, and the amount of proteins were evaluated with western blotting. (F) HA-tagged UBXN1 was expressed with or without NiV V in HEK293T cells. Then, the cells were treated with CHX, and the expression amount of HA-UBXN1 and GAPDH was quantitated as described in (E). The CHX assay was repeated three times, and the intensities of the bands were measured and summarized. Error bars indicate standard deviations (N = 3). **P < 0.01, ***P < 0.001, not significant (n.s.) on Student’s t test. The blots presented in (A–E) were cropped from different images to improve clarity. Full-length blots are presented in Supplementary Figure S2.
