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. 2002 Dec 1;30(23):5182–5192. doi: 10.1093/nar/gkf638

Figure 2.

Figure 2

In vitro arginine methylation of xCIRP2 protein by recombinant xPRMT1. (A) Physical interaction of xCIRP2 and xPRMT1 was analyzed by GST pull-down assay. One microgram of GST (lanes 1 and 2) or GST-xCIRP2 (lanes 3 and 4) was incubated with 2 µg of xPRMT1 in 100 µl of 20 mM Tris–HCl (pH 7.5), 100 mM NaCl without (lanes 1 and 3) or with (lanes 2 and 4) 80 µM AdoMet. Half of each reaction was analyzed by SDS–PAGE and the gel was stained with silver. One hundred nanograms of GST, GST-xCIRP2 and xPRMT1 proteins were electrophoresed in lanes 5, 6 and 7, respectively. (B) One microgram of recombinant HA-xCIRP2 protein (lanes 2, 4, 6 and 8) and 0.5 µg of xPRMT1 protein (lanes 3, 4, 7 and 8) were incubated in 20 µl of 20 mM Tris–HCl (pH 7.5) containing [3H]AdoMet. Proteins were separated by SDS–PAGE and stained with Coomassie Brilliant Blue (CBB staining). The gel was then dried and subjected to fluorography (Methylation). (C) One microgram of recombinant HA-xCIRP2 (lanes 1 and 5), HA-xCIRP2ΔRG4 (lanes 2 and 6), GST (lanes 3 and 7) and GST-RG4 (lanes 4 and 8) were methylated and visualized as in (B). (D) Schematic diagrams of recombinant xCIRP2 protein and its mutants used for in vitro methylation analysis. RNP1 and RNP2 are the conserved sequences in RRM (67).