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. 2019 Dec 2;10(12):903. doi: 10.1038/s41419-019-2147-3

Fig. 6. Arginine methylation of ATF4 by PRMT1 regulates ATF4 protein stability.

Fig. 6

a Immunoblot analysis for Flag-tagged ATF4-WT or R239K in HEK293T cells treated with cycloheximide (CHX, 10 μg/ml) for indicated hours. b The quantification of the relative Flag-ATF4 protein levels from experiments shown in panel (a). The signal intensities of Flag-ATF4 WT and R239K are normalized to β-actin loading control. The average values of WT at time point 0 is set to 1. Values are means of three independent determinants ±SEM. n = 3, *P < 0.05. ***P < 0.005. c Immunoblot analysis of ubiquitinated Flag-ATF4 protein in HEK293T cells transfected with expression vectors for Flag-ATF4 and ubiquitin along with control or PRMT1-HA. Cells were treated with control DMSO, Fura (10 μM) or DS-437 (50 μM) for 24 h. Four hour prior to harvest, cells were treated with MG132 (10 μM) to prevent proteasomal degradation. d Immunoblot analysis of ubiquitinated Flag-ATF4 proteins. HEK293T cells were cotransfected with expression vectors for ubiquitin in combination of ATF4 WT or R239K along with control or PRMT1-HA. Cells were treated with vehicle or a proteasome inhibitor MG132 (10 μM) for 4 h, followed by immunoprecipitation with Flag antibody and immunoblotting with Ub-K48 antibody to detect ubiquitinated ATF4 proteins.