Fig. 4
USP40 targeted CFLARL for de-ubiquitination. a A549 cells were seeded in 6-well plates. USP40 siRNA was transfected for 24 h and a non-sense siRNA was transfected as control. Cells were treated with CHX [10 μg/ml] and harvested at different time points (0, 4, 8, 12 h) for western blot analysis. The band intensity of CFLARL was quantified by Photoshop CS6 and plotted. Experiments were repeated three times, a representative experiment is presented. Every experimental group was compared with the negative control group. Error bars represent s.d. *P < 0.05. b, c H1299, A549 and H157 cells were seeded in 6-well plates. USP40 siRNA or plasmid were transfected for 24 h. Cells were treated with SAHA [2.0 μM] for 6 h and harvested for western blot analysis. d, e HEK293FT cells were prepared for GST pull down assays using GST-CFLARL, HIS-USP40 and HA-Ub plasmids or USP40 siRNA to detect the de-ubiquitination of USP40 targeted on CFLARL. f HA-Ub-K48WT and HA-Ub-K63WT were cloned from HA-Ub wild type plasmid. Only the 48th or 63rd lysine was natural while all other lysine amino acids were mutated to alanine. GST pull down assay was conducted in HEK293FT cells and the de-ubiquitination model of USP40 targeted on CFLARL was detected