Fig. 1.

GR associates with UPF1 and DCP1A via PNRC2. (A) IP experiments with endogenous GR. HEK293T cells were transiently cotransfected with plasmids expressing MYC-PNRC2 and FLAG-UPF1. Two days later, the cells were either treated or not treated with Dex for 3 h. Cell extracts were prepared and were either not treated or treated with RNase A for 15 min before IP. The IP experiments were performed using either α-GR antibody or a nonspecific mouse IgG (mIgG, control). The protein fractions before and after IPs were analyzed using Western blotting with the indicated antibodies. The levels of coimmunopurified MYC-PNRC2, FLAG-UPF1, and endogenous DCP1A were normalized to the level of immunopurified GR. The normalized levels obtained from IPs from the extracts of cells not treated with Dex were arbitrarily set to 1.0. For quantitative analysis, threefold serial dilutions of total cell extracts that were obtained before IP were loaded in the four leftmost lanes (Upper). Efficient digestion of cellular transcripts by RNase A treatment was demonstrated by the complete absence of cellular GAPDH mRNA according to RT-PCR using α-[³²P]-dATP and specific oligonucleotides. For quantitative analysis, total cell RNA samples obtained before IP were serially diluted twofold and analyzed using RT-PCR. The RT-PCR products were loaded in the four leftmost lanes (Lower). (B and C) IP experiments of endogenous GR from the extracts of cells depleted of PNRC2. The cells were transiently transfected with either PNRC2 siRNA or a nonspecific control siRNA. Two days later, the cells were retransfected with a plasmid expressing FLAG-UPF1. One day later, the cells were treated with Dex for 3 h before IP. After that, cell extracts were prepared and treated with RNase A before IP. (B) A Western blot of PNRC2 demonstrating specific down-regulation by siRNA. (C) IP experiments using either the α-GR antibody or mIgG (control). The levels of coimmunopurified FLAG-UPF1 and endogenous DCP1A were normalized to the level of immunopurified GR. The normalized levels obtained from IPs from the extracts of undepleted cells were arbitrarily set to 1.0. All data represent at least two independently performed transfection experiments, IPs, and RT-PCRs.