Fig. 6: Phosphorylation of JMJD3 by FGF21-activated PKA is critical for autophagy induction.

a Experimental outline (top) and spectrum from LC-MS/MS analysis identifying a JMJD3 peptide containing phosphorylated Thr-1044 (bottom). b PMH were transfected with plasmids as indicated, and after 48 h, were treated with vehicle or FGF21 (100 ng/ml) for 30 min. p-Thr-JMJD3 levels were detected by IP/IB and input protein by IB (n = 3 culture dishes). c Hepa1c1c7 cells were transfected with JMJD3 expression plasmids and treated with vehicle or FGF21 for 30 min. Flag-JMJD3 (green) was detected by immunofluorescence (scale bar = 5 μm). d PMH were transfected with JMJD3 expression plasmids as indicated and treated with vehicle or FGF21 for 30 min for CoIP (left) or 2 h for qRT-PCR (right). PPARα in flag-JMJD3 immunoprecipitates or in input detected by IB (left) and the mRNA levels of the indicated genes (right, n = 3 culture dishes). e FGF21-floxed or -LKO mice were fasted for 24 h or refed for 24 h after fasting. Levels of proteins were detected by IB (left) and p-Thr JMJD3 were detected by IP/IB (right) (n = 3 mice). f C57BL/6 mice were treated with 0.1 mg/kg FGF21 for 3 h and then, phosphorylated levels of PKA, ERK, and JMJD3 were detected by IB (n = 3 mice). g Immunoprecipitated flag-JMJD3 was incubated with ATP, PKA, or ERK1/2 as indicated and levels of p-Thr-JMJD3 were detected by IB. h Schematic of fragments of JMJD3 that were fused to GST (top). Binding of PKA or ERK1/2 to GST-JMJD3 proteins was detected by IB (bottom). i PMH were treated with FGF21 and with vehicle or a PKA (H89,10 μM) or MEK/ERK (PD98059, 40 μM) inhibitor for 30 min. Levels of p-Thr-JMJD3 were detected by IP/IB (top) and quantified (bottom, n = 4 culture dishes). Source data are provided as a Source Data file. d, i Values are presented as mean ± SD. Statistical significance was measured using (d, i) two-way ANOVA with the Bonferroni post-test. **P < 0.01, and NS statistically not significant.