Journal: Nature Communications

Article Title: Glycogen branching enzyme controls cellular iron homeostasis via Iron Regulatory Protein 1 and mitoNEET

doi: 10.1038/s41467-019-13237-8

Figure Lengend Snippet: Primer sequences.

Article Snippet: Human IRP1 cDNA (#HG10966-UT) and human GBE1 cDNA (#HG18919-UT) were acquired from Sino Biological Inc, while the human IRP2 cDNA (#OHS1770-202318020) was obtained from Dharmacon.

Techniques: Sequencing, Mutagenesis, CRISPR, Transfection, Construct, Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Glycogen branching enzyme controls cellular iron homeostasis via Iron Regulatory Protein 1 and mitoNEET

doi: 10.1038/s41467-019-13237-8

Figure Lengend Snippet: 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 ). 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. ) 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 w 1118 ) served as a negative control and calibrator (normalized expression = 1). SdhB mRNA harbours a validated IRE , . 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. ), 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.

Article Snippet: Human IRP1 cDNA (#HG10966-UT) and human GBE1 cDNA (#HG18919-UT) were acquired from Sino Biological Inc, while the human IRP2 cDNA (#OHS1770-202318020) was obtained from Dharmacon.

Techniques: Cotransfection, Immunoprecipitation, Western Blot, RNA Binding Assay, Negative Control, Knock-In, Expressing, Activity Assay, Mutagenesis, Standard Deviation, Staining, Transgenic Assay

Journal: Nature Communications

Article Title: Glycogen branching enzyme controls cellular iron homeostasis via Iron Regulatory Protein 1 and mitoNEET

doi: 10.1038/s41467-019-13237-8

Figure Lengend Snippet: IRP1 localizes to nuclei. a Protoporphyrin accumulation/autofluorescence in prothoracic glands (PG) from IRP1A -RNAi ( IRP1A IR ) flies reared on iron-depleted (=BPS) media for three generations. Adult survival relative to last larval stage (surviving third instar larvae = L3 = 100%). Scale bar = 250 μm. b Protoporphyrin accumulation/autofluorescence in PGs from IRP1A FCF animals (tissue-specific excision of IRP1A , Supplementary Fig. ) reared on iron-rich medium until L2, after which larvae were switched to BPS-supplemented food. Scale bar = 250 μm. c Survival of IRP1A and IRP1B null mutants (KO = knockout, Supplementary Fig. ). Error bars represent standard deviation from three biological replicates (each sample contained 50 individuals). d Subcellular localization of PG-specific, Flag-tagged IRP1A and IRP1B ( PG > IRP1A / PG > IRP1B transgenic lines, Supplementary Table ). DAPI was used to stain DNA/nuclei. Scale bar = 250 μm. e Subcellular localization of Flag-tagged transgenic human IRP1 and IRP2 ( PG > hIRP1 and PG > hIRP2 , Supplementary Table ) expressed specifically in the PG. Scale bar = 250 μm. f Subcellular localization of Flag-tagged proteins encoded by IRP1A 3F and IRP1B 3F knock-in alleles (Supplementary Fig. ) in control or AGBE mutant backgrounds ( AGBE +/+ = PG > FLP . AGBE −/− = PG > FLP ; AGBE FCM , Supplementary Fig. ). Scale bar = 500 μm. g Subcellular localization of Flag-tagged proteins encoded by IRP1A 3F and IRP1B 3F knock-in alleles (Supplementary Fig. ) in the fat body (FB) and salivary gland (SG). Scale bar = 500 μm. Source data are provided as a Source Data file.

Article Snippet: Human IRP1 cDNA (#HG10966-UT) and human GBE1 cDNA (#HG18919-UT) were acquired from Sino Biological Inc, while the human IRP2 cDNA (#OHS1770-202318020) was obtained from Dharmacon.

Techniques: Knock-Out, Standard Deviation, Transgenic Assay, Staining, Knock-In, Mutagenesis