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. 2015 Feb 24;290(15):9812–9822. doi: 10.1074/jbc.M114.616219

FIGURE 5.

FIGURE 5.

Residue Lys-131 of Pdx1 is methylated in cells. A, dot blot analysis using two dilutions of anti-Pdx1(Lys-131-Me) antibodies and methylated and unmethylated peptides corresponding to the N terminus of Pdx1 containing residue Lys-131. B, representative immunoblots (top panel) using anti-Pdx1(Lys-131-Me) antibody or anti-Pdx1 antibody on recombinant Pdx1 proteins methylated by Set7/9 in vitro. The quantitation of immunoblots (n = 3) is shown in the bottom panel. *, p < 0.05 compared with Pdx1 alone. C, HEK293 cells were transfected with Set7/9 and WT or mutant HA-tagged Pdx1 proteins, and then whole cell extracts were immunoprecipitated using anti-HA antibody, followed by immunoblot analysis using anti-Pdx1 and anti-Pdx1(Lys-131-Me) antibodies (top panel). The quantitation of methylated Pdx1 (relative to total immunoprecipitated Pdx1, n = 3) is shown in the bottom panel. *, p < 0.05 compared with WT Pdx1. D, top panel, chemical structure of the Lys demethylase inhibitor BP-107-7 (BP). Bottom panel, INS-1 832/13 β cells were treated with vehicle or BP-107-7 (10 μm) overnight, and then nuclear extracts were immunoprecipitated (IP) using anti-methyl Lys antibody followed by immunoblot using anti-Pdx1 antibody.