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. 2013 Jan 21;3:1090. doi: 10.1038/srep01090

Figure 4. The conserved GNHE motif homologous to protein phosphatase 1 is essential for debranching activity of hDbr1 in vitro.

Figure 4

A) Equal amount (0.1 μg each) of recombinant proteins purified from either Flag-vector- or the hDbr1 cDNA-transfected HEK293T cells on a Coomassie Brilliant Blue-stained 5–20% gradient SDS-polyacrylamide gel. The plasmids used for transfection are indicated above the panel as follows; Vector : Flag-pCDNA3 vector, WT : Flag-wild type hDbr1, H85A : Flag-hDbr1 containing histidine to alanine mutation at amino-acid position 85, H85S : Flag-hDbr1 harboring histidine to serine change at amino-acid position 85, N84A : Flag-hDbr1 carrying asparagine to alanine mutation at amino-acid position 84. Input lanes contain the lariat RNAs used for the assays. B) In vitro debranching assays with purified proteins shown in A). Lariat intron RNAs derived from either Ad2 (upper panel) and δ-crystallin (lower panel) pre-mRNA were incubated with recombinant proteins and analyzed by 6% denaturing polyacrylamide gel electrophoresis. The structure of RNAs corresponding to each band is demonstrated schematically. The asterisk shows the contaminated pre-mRNAs.