Journal: Scientific reports

Article Title: Assessment of the toxic effect of benzalkonium chloride on human limbal stem cells.

doi: 10.1038/s41598-025-96919-2

Figure Lengend Snippet: Fig. 3. (A) Agarose gel electropherogram of amplification products (passage 1). Lane 1: DNA ladder (M), lanes 2–3: KRT3 (101 bp), lanes 4–5: KRT12 (190 bp), lane 6: DNA ladder (M), lanes 7–8: TP63 (96 bp), lanes 9–10: ABCG2 (144 bp), lanes 11–12: ACTβ (131 bp), lane 13: DNA ladder (M). NC, negative control. (B) Visualization of lack of expression of CK3, and CK12 and expression of positive markers p63 and ABCG2, in LSCs cultured in a medium supplemented with H/I/E. FITC-conjugated specific antibodies (green); DAPI- stained cell nuclei (blue). IC, isotype control. Scale bars = 15 μm.

Article Snippet: After rinsing with PBS, the cells were incubated with blocking buffer for 1 h, rinsed again, and labelled with rabbit anti-ABCG2 polyclonal (1:50; 27286-AP; Proteintech, USA), rabbit anti-p63 polyclonal (1:50; 12143-1-AP; Proteintech, USA), mouse anti-CK3 monoclonal (1:50; ab68260; Abcam, UK), and rabbit anti-CK12 monoclonal (1:50; ab185627; Abcam, UK) primary antibodies at 4 °C overnight.

Techniques: Agarose Gel Electrophoresis, Amplification, Negative Control, Expressing, Cell Culture, Staining, Control

Journal: International Journal of Nanomedicine

Article Title: A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

doi: 10.2147/ijn.s58465

Figure Lengend Snippet: Figure 1 Docking model of p28 and p63 DBD. Notes: (A) The structure for the p28-p63 DBD complex was obtained from the best docking model. Blue: overall ribbon diagram of the p63 DBD, green: S4, S6, S7, S9, and S10 loops. (B) Ribbon diagram of the p63 DBD (yellow) superimposed on the electrostatic potential plot. p28 (green) binds to the hydrophobic region. (C) Relationship of p28 (green) and S4, S6, S7, S9, and S10 (orange) of the p63 DBD. (D) Amino acid sequence alignment of the DBD of p53 and p63, data from Chen et al.41 p28 binding residues are indicated in red on the aa sequence of p53 DBD (top) and p63 DBD (bottom). The Pirh2 binding motif on p53 is indicated by the blue bar. S, sheet structure; orange box, DNA-binding sites; green box, zinc finger sites. Abbreviation: DBD, DNA-binding domain.

Article Snippet: of p63 and its DBD Plasmid DNA carrying the human p63 gene was purchased from OriGene Technologies, Inc., (Rockville, MD, USA).

Techniques: Sequencing, Binding Assay

Journal: International Journal of Nanomedicine

Article Title: A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

doi: 10.2147/ijn.s58465

Figure Lengend Snippet: Figure 2 Tapping mode AFM images of protein monolayer. Notes: Tapping mode AFM images of: (A) p63 DBD monolayer immobilized on an amino-silane and glutaraldehyde functionalized glass slide; (B) p63 DBD monolayer after p28 deposition. All images were acquired in ambient air. Abbreviations: AFM, atomic force microscopy; DBD, DNA-binding domain.

Article Snippet: of p63 and its DBD Plasmid DNA carrying the human p63 gene was purchased from OriGene Technologies, Inc., (Rockville, MD, USA).

Techniques: Microscopy, Binding Assay

Journal: International Journal of Nanomedicine

Article Title: A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

doi: 10.2147/ijn.s58465

Figure Lengend Snippet: Figure 3 Schematic representation of AFS analysis. Notes: Left panel shows illustration of AFS analysis showing the p28 bound to the tip via a flexible linker and p63, p73, or p63 DBD immobilized on glass substrates. Right panel shows schematic representation of a force–piezo displacement cycle, showing a typical unbinding event. Abbreviations: AFS, atomic force spectroscopy; DBD, DNA-binding domain.

Article Snippet: of p63 and its DBD Plasmid DNA carrying the human p63 gene was purchased from OriGene Technologies, Inc., (Rockville, MD, USA).

Techniques: Force Spectroscopy, Binding Assay

Journal: International Journal of Nanomedicine

Article Title: A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

doi: 10.2147/ijn.s58465

Figure Lengend Snippet: Figure 6 Competitive immunoprecipitation assay for Cop1 and Pirh2. Notes: GST-p63 DBD and GST alone were immobilized on glutathione-Sepharose 4B beads and incubated in absence (−) or presence of p28 (+: 10, ++: 100 mole excess), followed by addition of MCF-7 lysates containing Cop1 and Pirh2. Samples were separated by SDS-PAGE and immunoblotted with either anti-Cop1 or anti- Pirh2 antibodies. Lysate: whole-cell lysates of MCF-7 used in assay stably expressed Cop1 and Pirh2. Numbers below Pirh2 bands are the relative percentage to the level of Pirh2 bound to p63 DBD in the absence of p28. Abbreviations: DBD, DNA-binding domain; GST, glutathione S-transferase; SDS- PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; IB, immunoblotting.

Article Snippet: of p63 and its DBD Plasmid DNA carrying the human p63 gene was purchased from OriGene Technologies, Inc., (Rockville, MD, USA).

Techniques: Immunoprecipitation, Incubation, SDS Page, Stable Transfection, Binding Assay, Polyacrylamide Gel Electrophoresis, Western Blot

Journal: International Journal of Nanomedicine

Article Title: A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

doi: 10.2147/ijn.s58465

Figure Lengend Snippet: Figure 5 Interaction of p28 with p53, p63, and p73. Notes: GST pull-down assay showing complex formation between GST-p28 and p53, p63, and p73. p53, p63, or p73 were detected with antibodies to anti-p53, p63, or p73. Whole-cell lysates (Lysate) from each cell line served as a positive control. The numbers indicated below each band are expressed relative to each control (Lysate) expressed as 100%. Abbreviations: GST, glutathione S-transferase; wt, wild-type; mut, mutant.

Article Snippet: of p63 and its DBD Plasmid DNA carrying the human p63 gene was purchased from OriGene Technologies, Inc., (Rockville, MD, USA).

Techniques: Pull Down Assay, Positive Control, Control, Mutagenesis

Journal: International Journal of Nanomedicine

Article Title: A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

doi: 10.2147/ijn.s58465

Figure Lengend Snippet: Figure 7 Effect of p28 on p63, p73, and E3 ligases on p53wt, mut breast cancer cells. Notes: MCF-7, MDD2, MDA-MB-231, and MCF-10A cells were treated with 50 µM p28 for 24–72 hours, and protein levels determined by immunoblotting (A). The expression of each gene was determined by RT-PCR (B). The numbers indicated below each band represent the level of expression relative to the control (control expressed as 100%). Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; wt, wild-type; mut, mutant; dom, dominant; neg, negative.

Article Snippet: of p63 and its DBD Plasmid DNA carrying the human p63 gene was purchased from OriGene Technologies, Inc., (Rockville, MD, USA).

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Reverse Transcription, Polymerase Chain Reaction, Mutagenesis

Journal: International Journal of Nanomedicine

Article Title: A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

doi: 10.2147/ijn.s58465

Figure Lengend Snippet: Figure 8 Effect of p28 on p63, p73, and E3 ligases on p53wt, mut melanoma cells. Notes: (A) Mel-29, Mel-23, and Mel-6 cells were treated with 50 µM p28 for 24–72 hours, and protein levels determined by immunoblotting. (B) The expression of each gene was determined by RT-PCR. The numbers below each band represent the level of expression relative to the control (control expressed as 100%). Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; wt, wild-type; mut, mutant.

Article Snippet: of p63 and its DBD Plasmid DNA carrying the human p63 gene was purchased from OriGene Technologies, Inc., (Rockville, MD, USA).

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Reverse Transcription, Polymerase Chain Reaction, Mutagenesis

Journal: Journal of breath research

Article Title: Influence of coexistence of mild OSA on airway mucus hypersecretion in patients with COPD.

doi: 10.1088/1752-7163/abd52e

Figure Lengend Snippet: Figure 2. Expression of MUC5AC (B) and TP63 (C) and the ratio of goblet to basal cells (D) in airway epithelial samples in patients with CTL, COPD, and OS (A) are shown. The scale bar represents 50 µm. Objective magnification: 200×. ns, P = 0.1234; ∗, P < 0.0332; ∗∗, P < 0.0021, ∗∗∗, P < 0.0002; ∗∗∗∗, P < 0.0001.

Article Snippet: Antibodies against MUC5AC (Abcam, ab24071, ab212636), TP63 (ABclonal, A2137), and β-tubulin (ABclonal, AC021) were used.

Techniques: Expressing

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

Journal: Frontiers in Immunology

Article Title: Retroductal dexamethasone administration promotes the recovery from obstructive and inflammatory salivary gland dysfunction

doi: 10.3389/fimmu.2024.1418703

Figure Lengend Snippet: Macrophages recruited by inflammatory cytokines exacerbated the fibrosis, which dexamethasone reduced. Immunohistochemistry staining was conducted to detect TNF (A) , IL-1β (C) , F4/80 (E) , and LY6G (G) . Images were quantified based on the staining intensity of positive areas. Quantifications of TNF (B) , IL-1β (D) , F4/80 (F) , and LY6G (H) . (I) MTC staining images were obtained using an Eclipse Ti–U2 inverted microscope, and the fibrotic area (yellow arrows) is indicated. (J) Fibrosis ratio was assessed using blue fibrotic areas after MTC staining. (K) IF staining was performed for epithelial (CDH1; green) and stromal (PDGFRB; red) cells, and (L) quantified positive areas for PDGFRB. Scale bar = 50 μm. N = 3~5 mice per group were used. The densitometric analysis involved 6~8 random field selections, with computations performed using Image J software. The IF quantification was expressed as a percentage by normalizing the positive area by the nuclei area. Data are presented as the mean ± SD. A one-way ANOVA with Tukey’s post hoc test was used to compare groups; * compared with Sham; # compared with Ligation; $ compared with Deligation+PBS. * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.05, ## p < 0.01, ### p < 0.001, $ p < 0.05, $$ p < 0.01, and $$$ p < 0.001.

Article Snippet: Primary antibodies against TNF (# NBP1–19532, 1:200; NOVUS, Centennial, CO, USA), IL-1β (# NB600–633,1:200; NOVUS), F4/80 (# A18637, 1:100; Abclonal, Woburn, MA, USA), and Ly-6G (# A20861, 1:50; Abclonal) were applied.

Techniques: Immunohistochemistry, Staining, Inverted Microscopy, Software, Ligation

Journal: Journal of Biological Chemistry

Article Title: Phosphorylated TP63 Induces Transcription of RPN13, Leading to NOS2 Protein Degradation

doi: 10.1074/jbc.m110.158642

Figure Lengend Snippet: FIGURE 1. Cisplatin induces RPN13 expression at the RNA level. Wild- type Np63 and Np63-S385G cells were treated with control medium () or 10 g/ml cisplatin (CIS; ) for 12 h. GAPDH was used as a loading control. A, RT-PCR analysis for RPN13 transcription. B, qPCR. Values for RPN13 (in relative units (RU)) were normalized to values for GAPDH, and values obtained from the control untreated samples were designated as 1. Experiments were performed in triplicate. FIGURE 2. Schematic representation of the human RPN13 gene pro- moter. The sequence of the human 1500-bp RPN13 promoter was found on the UCSC Genome Bioinformatics human genome web site, and certain potential TF-responsive elements (RE) were defined using TFSEARCH soft- ware. TF sequences are shown in boldface. The TSS is shown as an uppercase letter. The following responsive elements were located in the RPN13 pro- moter: TP63 (1376/1354, 1231/1216, 1189/1167, and 500/ 481), NF-Y/DDIT3 (1267/1246, 93/71, and 65/37), NF-B (995/ 985 and 724/716), STAT (857/843), and GAS (gamma-activated site; 971/862).

Article Snippet: Antibodies—We used a rabbit anti- Np63 polyclonal antibody (Ab-1, EMD Chemicals); a rabbit anti-TP63 (TP73L) monoclonal antibody (clone Y289, NB110-57309) and a rabbit anti-DDIT3 (DNA damage-inducible transcript 3) polyclonal antibody (NB100-78344) (Novus Biologicals); mouse monoclonal antibodies against -actin (Sigma) and NF-YA (Rockland Immunochemicals); and rabbit polyclonal antibodies against DNA topoisomerase II (TOP2A, ab74715), RPN13 (ab91567), NF- B p65 subunit (ab32536), NF- B p50 subunit (ab7549), STAT3 (signal transduction activator of transcription 3; ab32500), and UCH37 (ab38528) (Abcam).

Techniques: Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Sequencing