Figure 4: GnRH activates NSun2 by a PKA-dependent phosphorylation.

(a,b) LβT2 cells were treated by GnRH (10 nM for 4 h). (a) Nsun2, quantified by western blot analysis, was down-regulated by the GnRHa treatment (n = 10). (b) Nsun2 mRNA quantified by qRT-PCR was lower albeit non-significantly in GnRH-treated cells relative to control cells (n = 9). (c,d) Protein extracts were eluted from a phosphoprotein enrichment column and analysed by western blotting using an anti-NSun2 antibody. (c) Representative blot comparing input proteins from GnRH-treated or control cells with eluted proteins from enrichment columns. (d) LβT2 cells were either treated by GnRHa (10 nM for 4 h) or the cAMP analogue 8Br-cAMP or were pre-treated with the PKA inhibitor Rp-cAMP before the same GnRHa treatment. Activation of PKA using a 8Br-cAMP treatment had similar increasing effect as GnRHa on the phosphorylation state of NSun2 (n = 4). (e) The same treatment had similar increasing effect as GnRHa on the methylated fraction of miR-125b, whereas pre-treatment with the PKA inhibitor Rp-cAMP prevented this effect (n ≥ 4). (f) Same treatments had corresponding inverse effects on miR-125b cellular content (n ≥ 8). (g) LβT2 cells were either pre-treated with the PKA inhibitor H89 followed or not by a GnRHa treatment (10 nM for 4 h) or treated by GnRHa alone. The basal level of phosphorylation was not affected by the PKA inhibitor treatment alone. The GnRH-induced phosphorylation of NSun2 was prevented by the PKA inhibitor (n ≥ 5). (h) LβT2 cells were treated by GnRHa (10 nM for 10 min). Protein extracts were immunoprecipitated using anti-NSun2 antibody. Co-immunoprecipitated proteins were analysed by western blotting using anti-PRKACA. Representative blot comparing immunoprecipitated NSun2 proteins from GnRH-treated or control cells. The catalytic subunit of PKA was associated with NSun2 10 min after the GnRHa-treatment. Representative western blot images are given on Fig. S2. *P < 0.05; **P < 0.01; ***P < 0.001.