Fig. 2
GMEB1 enhanced the stability of CFLARL. a Relative mRNA levels of GMEB1 and CFLARL were determined by quantitative reverse transcription-polymerase chain reaction (q-PCR) in A549 cell line when the cell transfected with GMEB1 siRNA for 24 h. Error bars represent s.d. ***P < 0.001. b, c Calu-1 and A549 cells were seeded in 6-well plates. GMEB1 siRNA or plasmid were transfected for 24 h and a non-sense siRNA or empty vector was transfected as control. Cells were treated with cycloheximide (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. d, e A549, H1299, Calu-1 and H1792 cells were seeded in 6-well plates. GMEB1 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. f A549-LUC, A549-shGMEB1–1# and A549-shGMEB1–2# were seeded in 6-well plates. GMEB1 plasmid was transfected for 24 h. Cells were harvested for western blot analysis. g H1299 cells were seeded in 6-well plates. GMEB1 siRNA was transfected for 24 h. Cells were treated with DMSO, MG132 [20 μM] and E64D [15 μM] for 6 h. Cells were harvested for western blot analysis. h HEK293FT cells were prepared for GST pull down assay using GST-CFLARL, FLAG-GMEB1 and HA-Ub plasmids to detect the function of GMEB1 on the ubiquitination of CFLARL