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. 2015 May 21;11(5):e1005263. doi: 10.1371/journal.pgen.1005263

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© 2015 Choi et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Fig 3 — (A-B) Effects of the fat body-specific knockdown of HDAC4 (HDAC4 RNAi) on TAG amounts (A) and bmm gene expression (B) in LKB1 and SIK3 null mutants. Genotypes are as follows: FB> (FB-Gal4/+), LKB1^X5,FB> (FB-Gal4/+;LKB1 ^X5 /LKB1 ^X5), SIK3^Δ5–31,FB> (FB-Gal4,SIK3 ^Δ5–31 /SIK3 ^Δ5–31), FB>HDAC4 RNAi (FB-Gal4/+;UAS-HDAC4 RNAi/+), LKB1^X5,FB>HDAC4 RNAi (FB-Gal4/+;LKB1 ^X5 /LKB1 ^X5,UAS-HDAC4 RNAi), and SIK3^Δ5–31,FB>HDAC4 RNAi (FB-Gal4,SIK3 ^Δ5–31 /SIK3 ^Δ5–31 ;UAS-HDAC4 RNAi). (C) Drosophila HDAC4 protein structure showing three SIK3 phosphorylation sites and an HDAC class IIa domain (top panel). Immunoblot analyses showing the effect of wild-type and constitutively active (T196E) SIK3 on HDAC4 Ser239 phosphorylation in larvae (middle four panels). Densitometric analyses of phospho-HDAC4 bands (bottom panel). FB-Gal4 was used to drive transgene expression. Anti-phospho-Ser239 HDAC4, -FLAG (HDAC protein), -Myc (SIK3 protein), and -β-tubulin (TUB) antibodies were used. (D) Immunohistochemical analyses of HDAC4 (anti-FLAG antibody, green) in the fat body cells of wild type (first, second, and third rows), LKB1 mutant (LKB1 ^X5) (fourth row) and SIK3 mutant (SIK3 ^Δ5–31) (fifth row) L3 larvae in feeding or 4 hr fasting condition as denoted in figures. Similar experiments were also conducted for phosphorylation-defective and constitutively active HDAC4 (HDAC^3A) in wild type L3 larvae in feeding (sixth row) or 4 hr fasting condition (bottom row). Cell nuclei were stained by Hoechst 33258 (blue). The graphs on the right of each image showed the intensity plot profile for each antibody staining along the red lines. Genotypes are as follows: Control (FB-Gal4/+), FLAG-HDAC4 (FB-Gal4/UAS-HDAC4), FLAG-HDAC4^3A (FB-Gal4/UAS-HDAC4 3A), FLAG-HDAC4,LKB1^X5 (FB-Gal4/UAS-HDAC4;LKB1 ^X5/LKB1 ^X5), and FLAG-HDAC4,SIK3^Δ5–31 (FB-Gal4,SIK3 ^Δ5–31/UAS-HDAC4,SIK3 ^Δ5–31). Scale bars, 20 μm. (E) Effect of fat body-specific expression of constitutively active SIK3 (SIK3 T196E) on bmm gene expression in larvae expressing wild-type HDAC4 in the fat body. Genotypes are as follows: FB> (FB-Gal4/+), FB>HDAC4^WT (FB-Gal4/UAS-HDAC4), FB>SIK3^TE (FB-Gal4/+;UAS-SIK3 T196E/+), and FB>HDAC4 ^WT,SIK3^TE (FB-Gal4/UAS-HDAC4;UAS-SIK3 T196E/+). (F-G) Effects of the fat body-specific knockdown of bmm (bmm RNAi) on TAG amounts (F) and bmm gene expression (G) in LKB1 and SIK3 null mutants. Genotypes are as follows: FB> (FB-Gal4/+), LKB1^X5,FB> (FB-Gal4/+;LKB1 ^X5 /LKB1 ^X5), SIK3^Δ5–31,FB> (FB-Gal4,SIK3 ^Δ5–31 /SIK3 ^Δ5–31), FB>bmm RNAi (FB-Gal4/;UAS-bmm RNAi/+), LKB1^X5,FB>bmm RNAi (FB-Gal4/+;LKB1 ^X5 /LKB1 ^X5,UAS-bmm RNAi) and SIK3^Δ5–31,FB>bmm RNAi (FB-Gal4,SIK3 ^Δ5–31 /SIK3 ^Δ5–31 ;UAS-bmm RNAi). Data are presented as mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001).