Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2019 Nov 29;10:5463. doi: 10.1038/s41467-019-13237-8

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Fig. 2 — AGBE interacts with IRP1A. a Co-transfection of S2 cells with plasmids encoding Flag-tagged IRP1A variants (IRP1A^*) and Myc-tagged AGBE followed by immunoprecipitation via anti-Myc antibodies and Western blotting. Names shown in red indicate the protein used as bait. IRP1A: wild type IRP1A, IRP1A^C450S: constitutively RNA-binding IRP1A, IRP1A^3R3Q: non-RNA-binding form of IRP1A (Supplementary Table 1). Myc-tagged enhanced GFP (eGFP^M) served as a negative control. Input lane represents 10% of the sample. Presence of co-immunoprecipitated proteins were tested with anti-Flag antibodies. b Like A, but co-transfection of S2 cells with plasmids encoding Flag-tagged human IRP1 (aka Aco1) and Myc-tagged human GBE1, as well as eGFP^M as a negative control. c Quantitative RNA-immunoprecipitation (RIP). Samples from larvae carrying Flag-tagged knock-in alleles of IRP1A (IRP1A^3F, IRP1A^C450S.3F, and IRP1A^3R3Q.3F) (Supplementary Fig. 4) were normalized via Western blotting to visualize Flag-tagged proteins followed by ImageJ quantification. Western blot of adjusted samples shown below graph. Untagged IRP1A (control line w¹¹¹⁸) served as a negative control and calibrator (normalized expression = 1). SdhB mRNA harbours a validated IRE^72,73. Co-immunoprecipitated SdhB mRNA was quantified via qPCR. Error bars represent 95% confidence intervals from three biological replicates. ***p < 0.001, **p < 0.01, *p < 0.05. d Aconitase activity. Same IRP1A alleles and normalization procedure as described in c, except that IRP1A^3F served as the control (normalized to 1). All alleles were crossed into an IRP1B^−/− mutant background to eliminate the aconitase activity of IRP1B. Further, we removed mitochondria via ultracentrifugation to reduce the contribution of mitochondrial aconitase. Error bars represent standard deviation from three biological replicates. ** p < 0.01, *p < 0.05. e Survival rates of PG > FLP; AGBE^FCM animals (Supplementary Fig. 4), which causes Flippase-mediated excision of the AGBE transcription unit specifically in the prothoracic gland (PG). Tested in either the presence or absence of the IRP1A and IRP1A^C450S transgenes that are also expressed in a PG-specific manner. Error bars represent standard deviation from three biological replicates (each sample contained 50 individuals). f Larval and adult phenotypes of PG > FLP; AGBE^FCM and PG > Nos^IR-X animals expressing IRP1A^C450S or wild type IRP1A transgenes. Arrows point to red-stained PG. g Ring glands dissected from PG > AGBE^IR1 larvae in the presence or absence of the following transgenic cDNAs: IRP1A (wild type IRP1A); IRP1A^C450S (constitutively RNA-binding); IRP1A^3R3Q (non-RNA-binding); YAco1^WT: wild type yeast aconitase (mitochondrial); YAco1^∆Sp (cytoplasmic); hIRP1 & hIRP2: human IRP1 & IRP2. Scale bar = 250 μm. All transgenes are expressed in a PG-specific manner via the Gal4-UAS system. Source data are provided as a Source Data file.