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Fig. 4 | Journal of Experimental & Clinical Cancer Research

Fig. 4

From: The arginine methyltransferase PRMT5 and PRMT1 distinctly regulate the degradation of anti-apoptotic protein CFLARL in human lung cancer cells

Fig. 4

PRMT5 and PRMT1 regulate the polyubiquitination of CFLARL by affecting the interaction between CFLARL and ITCH. a H1299 cells were seeded into a 6-cm cell culture plate and transfected with PRMT5 siRNAs on the second day. After 24 h, the cells were seeded again into a 6-well cell culture plate to ensure that each well contained identical cell densities. Then, the cells were treated with 20 μmol/L MG132 for 4 h or 15 μmol/L E64D for 6 h. The cells were harvested for the preparation of the whole-cell protein lysates and subsequent western blot analysis. b H1299 cells were seeded into a 6-cm cell culture plate and transfected with the PRMT1 plasmid on the second day. After 24 h, the cells were seeded again into a 6-well cell culture plate to ensure that each well contained identical cell densities. Then, the cells were treated with 20 μmol/L MG132 for 4 h or 15 μmol/L E64D for 6 h. The cells were harvested for the preparation of the whole-cell protein lysates and subsequent western blot analysis. c Cells were transfected with the pcDNA3.1-FLAG-CFLARL or pcDNA3.1-HA-UB plasmids and co-transfected with ctrl siRNA or PRMT5 siRNA. The cells were harvested and prepared for the IP assay after 16 h. Then, the cells were treated with 20 μmol/L MG132 for 4 h. The polyubiquitination level of the CFLARL protein was detected by western blotting. d Cells were transfected with the pcDNA3.1-FLAG-CFLARL or pcDNA3.1-HA-UB plasmids and co-transfected with ctrl siRNA or PRMT1 siRNA. The cells were harvested and prepared for the IP assay after 16 h, and then, the cells were treated with 20 μmol/L MG132 for 4 h. The polyubiquitination level of the CFLARL protein was detected by western blotting. e Cells were transfected with the pcDNA3.1-FLAG-CFLARL /pcDNA3.1-HA-ITCH plasmids and co-transfected with pcDNA3.1 or pcDNA3.1-MYC-PRMT5. The cells were harvested and prepared for the co-IP assay after 24 h. The protein levels of CFLARL and HA were detected by western blotting. F HEK293FT cells were transfected with the pEBG-GST-CFLARL/ pcDNA3.1-HA-ITCH plasmids and co-transfected with pcDNA3.1 or pcDNA3.1-MYC-PRMT1. The cells were harvested and prepared for the GST pull-down assay after 24 h. The protein levels of CFLARL and ITCH were detected by western blotting. ACTB or GAPDH expression was detected as a loading control

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