Figure 6.
Double-SCR of MTCH2 reduces the stability of the protein generated. A and B, Western blotting of lysates of HEK293 cells transfected with plasmids expressing FLAG-HA-tagged MTCH2 isoforms. RT-PCR results show comparable expression of all three constructs at the RNA level. In B, cells were treated with 10 μm MG132 for 5 h. Assays were done 24 h after transfection. C, t1/2 of FLAG-HA–tagged MTCH2 isoforms. HEK293 cells transfected with plasmids expressing MTCH2 isoforms were treated with 100 µg/ml of CHX for the indicated duration and their lysates were used for Western blot to check the expression of FLAG-HA–tagged isoforms. D, ISR2 sequence was cloned downstream of and in-frame with the coding sequence of GFP such that ISR2 is translated along with GFP. Western blotting and RT-PCR analyses of GFP expression with or without ISR2 or with a stop codon between them, in transfected HEK293 cells are shown. E, fluorescence microscopy images and flow cytometry analyses (mean ± S.D., n = 3) of HEK293 cells transfected with GFP-expressing constructs described in D, scale bar, 50 μm. Two-tailed Student's t test was used to calculate the p value. F, Western blotting image showing increased expression of GFP-ISR2 in cells treated with MG132.