Journal: ACS chemical biology

Article Title: Conditional covalent lethality driven by oncometabolite accumulation

doi: 10.1021/acschembio.2c00384

Figure Lengend Snippet: (a) Structure of MP-1 clickable probe. (b) Dose-dependent labeling of proteomes by clickable probe. Concentrations: 0, 12.5, 25, 50, 75, 100 μM. (c) Schematic for validating target labeling by MP-1 chemotype. (d) Evaluation of putative MP-1 target enrichment by clickable probe following proteomic incubation (0, 10, 50, 100, 200 and 500 μM, 12 h), click chemistry (biotin-N3), streptavidin enrichment, and immunoblotted for TRMT1, Phosphofructokinase platelet type (PFKP) and Cytosolic iron-sulfur cluster assembly factor NUBP2. Ponceau stain image indicates protein loading for each lane. Western blot procedure described in general methods found in supplementary information. Briefly, following chemiluminescence analysis of TRMT1 the nitrocellulose membrane was stripped and re-probed for PFKP and NUBP2.

Article Snippet: TRMT1 (sc-373687), PFKP (sc-514824) and NUBP2 (sc-376784) antibodies were purchased from Santa Cruz Biotechnologies.

Techniques: Labeling, Incubation, Staining, Western Blot, Membrane

Journal: Frontiers in Oncology

Article Title: Serum autoantibody profiling of oral squamous cell carcinoma patients reveals NUBP2 as a potential diagnostic marker

doi: 10.3389/fonc.2023.1167691

Figure Lengend Snippet: Increased levels (Normalized RFU) of protein autoantibodies in serum samples from patients with oral squamous cell carcinoma (OSCC)as compared to those from healthy volunteers (Control) for (A) GGPS1, (B) KRAS, (C) MAP2K6, (D) NUBP2, (E) NSBP1, (F) PRDX1, (G) PSME3, (H) PTPN20A, (I) PYCR1, (J) RPA2, (K) S100A9, (L) TK1, (M) TSPY2, (N) TSPY3, (O) XAGE4. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Article Snippet: The mouse monoclonal anti-NUBP2 antibody was purchased from Santa Cruz Biotechnology Inc. (sc-376784) and used at 1:50 dilution.

Techniques: Control

Journal: Frontiers in Oncology

Article Title: Serum autoantibody profiling of oral squamous cell carcinoma patients reveals NUBP2 as a potential diagnostic marker

doi: 10.3389/fonc.2023.1167691

Figure Lengend Snippet: Details of autoantibodies increased in OSCC serum as compared to controls.

Article Snippet: The mouse monoclonal anti-NUBP2 antibody was purchased from Santa Cruz Biotechnology Inc. (sc-376784) and used at 1:50 dilution.

Techniques: Binding Assay, Expressing, Amplification, Over Expression, Phospho-proteomics

Journal: Frontiers in Oncology

Article Title: Serum autoantibody profiling of oral squamous cell carcinoma patients reveals NUBP2 as a potential diagnostic marker

doi: 10.3389/fonc.2023.1167691

Figure Lengend Snippet: Representative immunohistochemistry images showing positive staining of NUBP2 in Oral squamous cell carcinoma. Parietal cells were used as positive controls.

Article Snippet: The mouse monoclonal anti-NUBP2 antibody was purchased from Santa Cruz Biotechnology Inc. (sc-376784) and used at 1:50 dilution.

Techniques: Immunohistochemistry, Staining

Journal: Frontiers in Oncology

Article Title: Serum autoantibody profiling of oral squamous cell carcinoma patients reveals NUBP2 as a potential diagnostic marker

doi: 10.3389/fonc.2023.1167691

Figure Lengend Snippet: Immunohistochemical results for NUBP2.

Article Snippet: The mouse monoclonal anti-NUBP2 antibody was purchased from Santa Cruz Biotechnology Inc. (sc-376784) and used at 1:50 dilution.

Techniques: Immunohistochemical staining, Staining

Journal: Frontiers in Oncology

Article Title: Serum autoantibody profiling of oral squamous cell carcinoma patients reveals NUBP2 as a potential diagnostic marker

doi: 10.3389/fonc.2023.1167691

Figure Lengend Snippet: (A) NUBP2 expression across sample types in GDC TCGA Head and Neck Cancer RNASeq-HTSeq data from Xena Functional Genomics Explorer. (B) NUBP2 expression across clinical stages in GDC TCGA Head and Neck Cancer RNASeq-HTSeq data from Xena Functional Genomics Explorer. Kaplan Meier plots showing correlation of NUBP2 expression with (C) Overall Survival and (D) Disease Free survival in patients with HNSCC from TCGA. (E) Figure showing sequence similarity of NUBP2 across various species. (F) Conservation of NUBP2 across species.

Article Snippet: The mouse monoclonal anti-NUBP2 antibody was purchased from Santa Cruz Biotechnology Inc. (sc-376784) and used at 1:50 dilution.

Techniques: Expressing, Functional Assay, Sequencing

Journal: PLoS ONE

Article Title: Nubp1 Is Required for Lung Branching Morphogenesis and Distal Progenitor Cell Survival in Mice

doi: 10.1371/journal.pone.0044871

Figure Lengend Snippet: (A–E) Nubp1 and its homolog Nubp2 are expressed in the distal lung epithelium. (A, C) In situ hybridization of Nubp1 and Nubp2 on E11.5 lung whole mounts. (B, D) Q-PCR for Nubp1 and Nubp2 at different developmental stages reveals no expression differences in mutant versus wild-type lungs. (E) PCR amplification of the indicated genes on FACS sorted E18.5 distal lung epithelial cells (GFP +) versus remaining GFP (-) cells. (F) Nubp2 interacts with Nubp1 but not with the mutant protein (Nubp1-m1Nisw). A549 cells overexpressing the indicated genes were processed for immunoprecipitation and analyzed by western blotting. (G–H) Mutation in the Nubp1 gene (Nubp1-m1Nisw) does not alter its cellular localization. A549 cells were stably transfected with GFP-Nubp1 or GFP-Nubp1(m1Nisw) and processed for immunofluorescence.

Article Snippet: For immunoprecipitation, cell lysates were incubated with 1 ug of anti-GFP antibody (Sigma, 1∶1000 dilution) and western blotting was performed with anti-myc or anti-Nubp2 antibody (Sigma, 1∶1000 dilution).

Techniques: In Situ Hybridization, Expressing, Mutagenesis, Amplification, Immunoprecipitation, Western Blot, Stable Transfection, Transfection, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Iron-regulated assembly of the cytosolic iron–sulfur cluster biogenesis machinery

doi: 10.1016/j.jbc.2022.102094

Figure Lengend Snippet: CIA components and substrates form higher order complexes . A , HEK293 cells were treated with 100 μg/ml ammonium ferric citrate (FAC) or 100 μM deferoxamine (DFO) for 8 h to create an iron-sufficient or -deficient environment, respectively. Whole cell extracts (WCEs) were resolved by SDS-PAGE and blotted with antibodies against known components of the cytosolic iron–sulfur cluster assembly (CIA) pathway, CIA substrates, loading control α-tubulin, FAC treatment control FBXL5, and DFO treatment control IREB2. B , Flp-In T-Rex 293 cell line (Flp-In 293) engineered to stably express NUBP2 was induced overnight with 1 μg/ml doxycycline. Affinity purified NUBP2 and associated proteins were identified by bottom-up proteomics. Data-dependent acquisition of two technical replicates was performed. Spectra count (SpC) for selected proteins combined from the two technical replicates was shown. C , parallel reaction monitoring was conducted on HEK293 whole cell lysate with indicated amounts of protein using a targeted proteomics assay that monitors the abundance of known CIA factors (ABCB7, GLRX3, BOLA2, CIAPIN1, NUBP1, NUBP2, CIAO3, CIAO1, CIAO2B, CIAO2A, MMS19) and prototypical substrates (CDKAL1, DNA2, ERCC2, POLD1, and ABCE1). Two technical replicates were acquired. Intensities were normalized for each precursor to the highest intensity in a replicate. D , Flp-In 293 cells that stably express NUBP2 were induced with 1 μg/ml doxycycline. Affinity purified NUBP2 and associated proteins were identified by acquisition of a targeted proteomic assay containing CIA scaffold complex components (NUBP1 and NUBP2), CIAO3, CIA targeting complex components (MMS19, CIAO1, and CIAO2B) and prototypical substrates (CDKAL1, DNA2, ERCC2, POLD1, and ABCE1). Two biological replicates were performed. Acquired spectra were searched with MaxQuant. Each edge represents a peptide identified. Solid edges connect bait protein to known interactors while dashed edges connect to novel interactors discovered in our study. Edge widths correspond to the posterior error probability of each peptide. E , Flp-In 293 background cells or cells expressing NUBP2 were either directly harvested or after treatment with 1% formaldehyde. Anti-HA immunoprecipitation were performed. WCEs and anti-HA immunoprecipitates were blotted for CIAO3, CIA targeting complex components, and the CIA substrate CDKAL1. CIAO1: probable cytosolic iron-sulfur protein assembly protein CIAO1; CIAO2B: cytosolic iron-sulfur assembly component 2B; CIAO3: cytosolic iron-sulfur assembly component 3; MMS19: MMS19 nucleotide excision repair protein homolog; NUBP1: nucleotide-binding protein 1; NUBP2: nucleotide-binding protein 2.

Article Snippet: Membranes were blocked with either 5% milk or 5% BSA before blotting with primary antibodies against CIAPIN1 (Santa Cruz Biotechnology sc-271298, Lot #H2317), GLRX3 (Santa Cruz Biotechnology sc-100601 Lot#C1811), CIAO3 (Santa Cruz Biotechnology sc-514078 Lot #K1914 or Sigma Aldrich SAB4502760), NUBP1 (Santa Cruz Biotechnology sc-514175, Lot #0715), NUBP2 (Proteintech 15409-1-AP), MMS19 (Proteintech 16015-1-AP or 66049-1-IG), CIAO1 (Cell Signaling 87027S Lot:1), CIAO2B (Proteintech 20108-1-AP), CIAO2A (Proteintech 20776-1-AP), ERCC2 (Santa Cruz Biotechnology sc-101174, Lot #K0414), FTH1 (Cell Signaling #3998), FLAG (Sigma #F1804 and Proteintech), ISCU1/2 (Proteintech), HIF1α (Bethyl A300–286A), IRP2 (Santa Cruz Biotechnology sc-33682, Lot #B1116), FBXL5 (BioLegend Clone 3F5G12G9 or 10F4H9D12), GAPDH (Proteintech HRP-60004), and TUBA (Proteintech).

Techniques: SDS Page, Control, Stable Transfection, Affinity Purification, Data-dependent acquisition, Targeted Proteomics, Proteomic Assay, Expressing, Immunoprecipitation, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Iron-regulated assembly of the cytosolic iron–sulfur cluster biogenesis machinery

doi: 10.1016/j.jbc.2022.102094

Figure Lengend Snippet: CIAO3 interactions are regulated by intracellular iron level in a time-dependent manner . A–C , Flp-In 293 stably expressing 3HA-3FLAG–tagged CIAO3 was induced with 1 μg/ml doxycycline for 24 h and treated with 100 μg/ml FAC or 100 μM DFO for 8 h before harvesting. Two biological replicates were performed for each experiment. A , affinity purified CIAO3 and associated proteins were characterized by shotgun proteomics and quantified by MS1 intensity-based label-free quantification. Abundance of co-eluted ( B ) CIA scaffold complex components (NUBP1 and NUBP2) and ( C ) CIA targeting complex components (MMS19, CIAO1, and CIAO2B) were monitored using a targeted proteomic assay. Log2 of calculated protein intensities (Log2Intensities) under FAC or DFO condition were plotted with mean ± SD. ∗ denotes p < 0.05. D , Flp-In 293 cells or Flp-In 293 expressing NUBP2 were induced with 1 μg/ml doxycycline for 24 h. Cells expressing 3HA-3FLAG-NUBP2 were treated with 100 μg/ml FAC, untreated or treated with 100 μM DFO for 8 h before harvesting. WCEs and anti-HA immunoprecipitates (IP: HA) were analyzed by immunoblotting with antibodies against FLAG, CIAO3, loading control α-tubulin, and FAC/DFO treatment control FTH1. E , Flp-In 293 background cells or cells stably expressing 3HA-3FLAG-CIAO3 were induced with 1 μg/ml doxycycline overnight, untreated, or treated with 100 μg/ml FAC or 100 μM DFO for hours indicated. WCEs and anti-HA immunoprecipitates (IP: HA) were blotted with antibodies against indicated proteins. CIAO3, cytosolic iron–sulfur assembly component 3; CIAO1: probable cytosolic iron-sulfur protein assembly protein CIAO1; CIAO2B: cytosolic iron-sulfur assembly component 2B; CIAO3: cytosolic iron-sulfur assembly component 3; DFO, deferoxamine; FAC, ferric ammonium citrate; MMS19: MMS19 nucleotide excision repair protein homolog; NUBP1: nucleotide-binding protein 1; NUBP2: nucleotide-binding protein 2; WCE, whole cell extract.

Article Snippet: Membranes were blocked with either 5% milk or 5% BSA before blotting with primary antibodies against CIAPIN1 (Santa Cruz Biotechnology sc-271298, Lot #H2317), GLRX3 (Santa Cruz Biotechnology sc-100601 Lot#C1811), CIAO3 (Santa Cruz Biotechnology sc-514078 Lot #K1914 or Sigma Aldrich SAB4502760), NUBP1 (Santa Cruz Biotechnology sc-514175, Lot #0715), NUBP2 (Proteintech 15409-1-AP), MMS19 (Proteintech 16015-1-AP or 66049-1-IG), CIAO1 (Cell Signaling 87027S Lot:1), CIAO2B (Proteintech 20108-1-AP), CIAO2A (Proteintech 20776-1-AP), ERCC2 (Santa Cruz Biotechnology sc-101174, Lot #K0414), FTH1 (Cell Signaling #3998), FLAG (Sigma #F1804 and Proteintech), ISCU1/2 (Proteintech), HIF1α (Bethyl A300–286A), IRP2 (Santa Cruz Biotechnology sc-33682, Lot #B1116), FBXL5 (BioLegend Clone 3F5G12G9 or 10F4H9D12), GAPDH (Proteintech HRP-60004), and TUBA (Proteintech).

Techniques: Stable Transfection, Expressing, Affinity Purification, Quantitative Proteomics, Proteomic Assay, Western Blot, Control, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Iron-regulated assembly of the cytosolic iron–sulfur cluster biogenesis machinery

doi: 10.1016/j.jbc.2022.102094

Figure Lengend Snippet: CIAO3 interactions are altered by changes in cellular redox state and requires functional mitochondrial Fe-S cluster biogenesis . A , Flp-In 293 cells expressing 3HA-3FLAG-CIAO3 were treated with tert-butyl hydroperoxide (tBHP) for 4 h to introduce reactive oxygen species. WCEs and anti-HA immunoprecipitates were blotted with antibodies against indicated proteins and loading control GAPDH. B , Flp-In 293 cells expressing 3HA-3FLAG-CIAO3 or control cells were induced with 1 μg/ml doxycycline overnight and cultured in 21% O 2 or 1% O 2 for 16 h before harvesting. WCEs and anti-HA immunoprecipitates were immunoblotted with antibodies against NUBP2, loading control α-tubulin, and hypoxia treatment control HIF1α. C , mitochondrial iron-sulfur cluster biogenesis was disrupted by using siRNA to silence ISCU1/2 in Flp-In 293 cells for 48 h while control cells were treated with nontarget siRNA. Doxycycline was added to induce expression of 3HA-3FLAG-CIAO3. WCEs and HA immunoprecipitates were blotted with indicated antibodies. D and E , quantification of ( C ) by densitometry. Protein abundance of coimmunoprecipitated proteins were normalized to the protein level of immunoprecipitated bait (3HA-3FLAG-CIAO3). Mean ± SD was plotted for n = 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01. CIAO3, cytosolic iron–sulfur assembly component 3; CIAO1: probable cytosolic iron-sulfur protein assembly protein CIAO1; CIAO2B: cytosolic iron-sulfur assembly component 2B; CIAO3: cytosolic iron-sulfur assembly component 3; Fe-S, iron–sulfur; MMS19: MMS19 nucleotide excision repair protein homolog; NUBP1: nucleotide-binding protein 1; NUBP2: nucleotide-binding protein 2; WCE, whole cell extract.

Article Snippet: Membranes were blocked with either 5% milk or 5% BSA before blotting with primary antibodies against CIAPIN1 (Santa Cruz Biotechnology sc-271298, Lot #H2317), GLRX3 (Santa Cruz Biotechnology sc-100601 Lot#C1811), CIAO3 (Santa Cruz Biotechnology sc-514078 Lot #K1914 or Sigma Aldrich SAB4502760), NUBP1 (Santa Cruz Biotechnology sc-514175, Lot #0715), NUBP2 (Proteintech 15409-1-AP), MMS19 (Proteintech 16015-1-AP or 66049-1-IG), CIAO1 (Cell Signaling 87027S Lot:1), CIAO2B (Proteintech 20108-1-AP), CIAO2A (Proteintech 20776-1-AP), ERCC2 (Santa Cruz Biotechnology sc-101174, Lot #K0414), FTH1 (Cell Signaling #3998), FLAG (Sigma #F1804 and Proteintech), ISCU1/2 (Proteintech), HIF1α (Bethyl A300–286A), IRP2 (Santa Cruz Biotechnology sc-33682, Lot #B1116), FBXL5 (BioLegend Clone 3F5G12G9 or 10F4H9D12), GAPDH (Proteintech HRP-60004), and TUBA (Proteintech).

Techniques: Functional Assay, Expressing, Introduce, Control, Cell Culture, Quantitative Proteomics, Immunoprecipitation, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Iron-regulated assembly of the cytosolic iron–sulfur cluster biogenesis machinery

doi: 10.1016/j.jbc.2022.102094

Figure Lengend Snippet: Fe-S cluster incorporation into CIAO3 controls its interactions. A , schematic representation of Fe-S incorporation in wildtype CIAO3 and mutants. B – D , Flp-In 293 cells that stably express wildtype or mutant CIAO3 were induced with 1 μg/ml doxycycline for 24 h before harvesting. CIAO3 and associated proteins were affinity purified. Two biological replicates were analyzed. Protein abundances were monitored by a targeted proteomic assay containing known CIA components and a subset of prototypical CIA substrates after normalization to CIAO3 levels. Log2 transformed abundance of the co-eluted proteins (Log2Intensiteis) were plotted for the CIA scaffold complex ( B ), the CIA targeting complex ( C ), and selected CIA substrates ( D ). Mean ± SD was indicated. Protein levels of interactors co-eluted with mutant CIAO3 were compared to wildtype. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001. E , Flp-In 293 control cells, cells expressing wildtype 3HA-3FLAG-ERCC2 and cells expressing mutant ERCC2 lacking the MMS19 binding region were induced with 1 μg/ml doxycycline for 24 h. Cell lysates were immunoprecipitated. WCEs and HA immunoprecipitates were blotted with indicated antibodies. CIA, cytosolic iron–sulfur cluster assembly; CIAO3, cytosolic iron–sulfur assembly component 3; CIAO1: probable cytosolic iron-sulfur protein assembly protein CIAO1; CIAO2B: cytosolic iron-sulfur assembly component 2B; CIAO3: cytosolic iron-sulfur assembly component 3; Fe-S, iron–sulfur; MMS19: MMS19 nucleotide excision repair protein homolog; NUBP1: nucleotide-binding protein 1; NUBP2: nucleotide-binding protein 2; WCE, whole cell extract.

Article Snippet: Membranes were blocked with either 5% milk or 5% BSA before blotting with primary antibodies against CIAPIN1 (Santa Cruz Biotechnology sc-271298, Lot #H2317), GLRX3 (Santa Cruz Biotechnology sc-100601 Lot#C1811), CIAO3 (Santa Cruz Biotechnology sc-514078 Lot #K1914 or Sigma Aldrich SAB4502760), NUBP1 (Santa Cruz Biotechnology sc-514175, Lot #0715), NUBP2 (Proteintech 15409-1-AP), MMS19 (Proteintech 16015-1-AP or 66049-1-IG), CIAO1 (Cell Signaling 87027S Lot:1), CIAO2B (Proteintech 20108-1-AP), CIAO2A (Proteintech 20776-1-AP), ERCC2 (Santa Cruz Biotechnology sc-101174, Lot #K0414), FTH1 (Cell Signaling #3998), FLAG (Sigma #F1804 and Proteintech), ISCU1/2 (Proteintech), HIF1α (Bethyl A300–286A), IRP2 (Santa Cruz Biotechnology sc-33682, Lot #B1116), FBXL5 (BioLegend Clone 3F5G12G9 or 10F4H9D12), GAPDH (Proteintech HRP-60004), and TUBA (Proteintech).

Techniques: Stable Transfection, Mutagenesis, Affinity Purification, Proteomic Assay, Transformation Assay, Control, Expressing, Binding Assay, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Iron-regulated assembly of the cytosolic iron–sulfur cluster biogenesis machinery

doi: 10.1016/j.jbc.2022.102094

Figure Lengend Snippet: CIAO3 mutant associated with PAVMs cannot assemble into the higher order complexes . A , homology modeled human CIAO3 from iron-only hydrogenase (1FEH) showing distances from serine 161 to proline or to the C-terminal Fe-S cluster. B , sequence alignment of CIAO3 orthologs. C , WCEs and HA immunoprecipitates from Flp-In 293 cells expressing wildtype CIAO3, the S161I mutant, or control cells were analyzed by SDS-PAGE and immunoblotted with antibodies indicated. D – F , quantification of ( C ) by densitometry showing baseline corrected protein abundance of coimmunoprecipitated proteins with respect to the bait protein (3HA-3FLAG-CIAO3 wild-type or S161I). Mean ± SD was plotted for n = 3 independent experiments. ∗ p < 0.05. CIAO3, cytosolic iron–sulfur assembly component 3; CIAO1: probable cytosolic iron-sulfur protein assembly protein CIAO1; CIAO2B: cytosolic iron-sulfur assembly component 2B; CIAO3: cytosolic iron-sulfur assembly component 3; MMS19: MMS19 nucleotide excision repair protein homolog; NUBP1: nucleotide-binding protein 1; NUBP2: nucleotide-binding protein 2; PAVMS, pulmonary arteriovenous malformations; WCE, whole cell extract; Fe-S, iron–sulfur.

Article Snippet: Membranes were blocked with either 5% milk or 5% BSA before blotting with primary antibodies against CIAPIN1 (Santa Cruz Biotechnology sc-271298, Lot #H2317), GLRX3 (Santa Cruz Biotechnology sc-100601 Lot#C1811), CIAO3 (Santa Cruz Biotechnology sc-514078 Lot #K1914 or Sigma Aldrich SAB4502760), NUBP1 (Santa Cruz Biotechnology sc-514175, Lot #0715), NUBP2 (Proteintech 15409-1-AP), MMS19 (Proteintech 16015-1-AP or 66049-1-IG), CIAO1 (Cell Signaling 87027S Lot:1), CIAO2B (Proteintech 20108-1-AP), CIAO2A (Proteintech 20776-1-AP), ERCC2 (Santa Cruz Biotechnology sc-101174, Lot #K0414), FTH1 (Cell Signaling #3998), FLAG (Sigma #F1804 and Proteintech), ISCU1/2 (Proteintech), HIF1α (Bethyl A300–286A), IRP2 (Santa Cruz Biotechnology sc-33682, Lot #B1116), FBXL5 (BioLegend Clone 3F5G12G9 or 10F4H9D12), GAPDH (Proteintech HRP-60004), and TUBA (Proteintech).

Techniques: Mutagenesis, Sequencing, Expressing, Control, SDS Page, Quantitative Proteomics, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Iron-regulated assembly of the cytosolic iron–sulfur cluster biogenesis machinery

doi: 10.1016/j.jbc.2022.102094

Figure Lengend Snippet: Model for the regulated assembly of the CIA machinery in response to ROS, O 2 tension, intracellular iron levels, and Fe-S cluster assembly on CIAO3 . CIA, cytosolic iron–sulfur cluster assembly; CIAO3, cytosolic iron–sulfur assembly component 3; CIAO1: probable cytosolic iron-sulfur protein assembly protein CIAO1; CIAO2B: cytosolic iron-sulfur assembly component 2B; CIAO3: cytosolic iron-sulfur assembly component 3; Fe-S, iron–sulfur; MMS19: MMS19 nucleotide excision repair protein homolog; NUBP1: nucleotide-binding protein 1; NUBP2: nucleotide-binding protein 2; ROS: reactive oxygen species.

Article Snippet: Membranes were blocked with either 5% milk or 5% BSA before blotting with primary antibodies against CIAPIN1 (Santa Cruz Biotechnology sc-271298, Lot #H2317), GLRX3 (Santa Cruz Biotechnology sc-100601 Lot#C1811), CIAO3 (Santa Cruz Biotechnology sc-514078 Lot #K1914 or Sigma Aldrich SAB4502760), NUBP1 (Santa Cruz Biotechnology sc-514175, Lot #0715), NUBP2 (Proteintech 15409-1-AP), MMS19 (Proteintech 16015-1-AP or 66049-1-IG), CIAO1 (Cell Signaling 87027S Lot:1), CIAO2B (Proteintech 20108-1-AP), CIAO2A (Proteintech 20776-1-AP), ERCC2 (Santa Cruz Biotechnology sc-101174, Lot #K0414), FTH1 (Cell Signaling #3998), FLAG (Sigma #F1804 and Proteintech), ISCU1/2 (Proteintech), HIF1α (Bethyl A300–286A), IRP2 (Santa Cruz Biotechnology sc-33682, Lot #B1116), FBXL5 (BioLegend Clone 3F5G12G9 or 10F4H9D12), GAPDH (Proteintech HRP-60004), and TUBA (Proteintech).

Techniques: Binding Assay