Journal: Cell death & disease

Article Title: Mitochondrial angiotensin receptors in dopaminergic neurons. Role in cell protection and aging-related vulnerability to neurodegeneration.

doi: 10.1038/cddis.2016.327

Figure Lengend Snippet: Figure 1 Specificity of angiotensin receptor antibodies and localization of angiotensin receptors in mitochondria of dopaminergic neurons. (a) The expression of major RAS components in dopaminergic neurons was confirmed by RT-PCR and laser microdissection of dopaminergic neurons retrogradly labeled by intrastriatal injection of fluorescent red retrobeads (RRB). SN section showing labeled dopaminergic neurons before and after laser microdissection for RT-PCR. Expression of TH, AGT, AT1, AT2 and β-actin mRNA in laser-microdissected dopaminergic neurons (right) and homogenates of SN used as a positive control (left) are also shown. (b) Western blot densitometric bands corresponding to 10 μg of AT1 or AT2 receptor overexpression lysate containing a C-terminal DDKepitope tag (DYKDDDDK) fused (left), and 10 μg of empty vector transfected control cell lysate HEK293 (EV, right). A band of 45 kDa was detected with the AT1 antibody, while a band of 50 kDa was detected with the AT2 antibody. A monoclonal antibody against DDK detected the corresponding band in the protein lysates. Colocalization of mitochondria and AT1 and AT2 receptors in primary cultures of the nigral region (c) and neurons from the MES 23.5 dopaminergic cell line (d). Electron microscopy of AT1 and AT2 labeling in a neuronal process (e) and cytoplasm (f) of a dopaminergic neuron. Immunolabeling for AT1 was observed in the outer membrane and cristae (white arrows) of mitochondria. In addition, strong AT1 labeling was also seen in clusters of free ribosomes (empty arrowheads). Immunolabeling for AT2 was present in mitochondrial membranes (white arrows), in the rough endoplasmic reticulum (black arrowheads) and in small clusters of free ribosomes, some of which were in close proximity to labeled mitochondria (empty arrowheads). AGT, angiotensinogen; DA, dopaminergic; MTDR, MitoTracker Deep Red; SN, substantia nigra; TH, tyrosine hydroxylase. Scale bars: (a) 50 and 200 μm (SN section); (c) 5 μm; (d) 10 μm; (e and f) 500 nm

Article Snippet: The specificity of the antibodies used for WB and immunolabeling studies was established in previous studies: AT1 sc-3118135 and AT2 sc-9040.36,37 In addition, the specificity of the antibodies was confirmed in our laboratory by preadsortion with the corresponding synthetic peptide antigen.38 In the present study, we also used WB analysis of lysates from HEK293 cells transfected with AT1 or AT2 tagged to fusion tail DDK (TA50011 from Origene, Rockville, MD, USA; DDK tag: DYKDDDDK).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Laser Capture Microdissection, Labeling, Injection, Positive Control, Western Blot, Over Expression, Plasmid Preparation, Transfection, Control, Electron Microscopy, Immunolabeling, Membrane

Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: CIB1 forms a complex with EVER1 and EVER2. (A) CIB1 protein levels in LCLs derived from patients with loss-of-function mutations of EVER1 (D576*) and EVER2 (D362* or T150Mfs*3), and comparison with those in heterozygous carriers, positive controls (ctrl 1 and 2), P4 and P11, and patients with RHOH and MST1 deficiencies. (B) CIB1, EVER1, and EVER2 mRNA levels were assessed by RT-qPCR in controls ( n = 4), CIB1 m/m (P1–P6 and P12), EVER1 m/m, and EVER2 m/m ( n = 3 each) LCLs. Each symbol represents the mean of one cell line measured in three independent experiments. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison tests relative to healthy controls. (C) CIB1 protein levels in LCLs derived from patients with loss-of-function mutations of EVER1 (D576*), EVER2 (T150Mfs*3), and a healthy control after reconstitution with WT EVER1 or EVER2 by retroviral transduction and stable selection. Asterisk indicates a nonspecific band. (D) CIB1, EVER1, and EVER2 mRNA levels were measured by RT-qPCR in LCLs derived from patients with loss-of-function mutations of EVER1 (D576*), EVER2 (T150Mfs*3), or CIB1 and a healthy control after reconstitution with WT EVER1 or EVER2 by retroviral transduction and stable selection. The data were first normalized against RNaseP as a housekeeping gene and then against an appropriate untransduced parental cell line by the ΔΔCt method. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to the corresponding untransduced control. (B and D) ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) HEK293T cells were transfected with plasmids encoding CIB1-HA, FLAG-EVER1, and FLAG-EVER2 either separately or together; 24 h after transfection, samples were subjected to immunoprecipitation with FLAG (Fl)- or HA-specific antibodies (+). Samples incubated with nonspecific IgG served as specificity controls. Western blots were performed to detect coimmunoprecipitated CIB1-HA and FLAG-EVER1 or FLAG-EVER2. The immunoprecipitation of EVER1/2 and CIB1 was confirmed by reincubation with antibodies specific for FLAG and HA, respectively. The presence of all proteins was checked by Western blotting of an input sample taken before immunoprecipitation. GAPDH served as a loading control. vec, vector. (F) Healthy control keratinocytes were transfected with plasmids encoding CIB1-HA, FLAG-EVER1, and FLAG-EVER2 either alone or in combination; 24 h after transfection, cells were subjected to immunofluorescence imaging with Alexa Fluor 568–HA and Alexa Fluor 488–FLAG antibody combinations. DAPI was used for counterstaining. Colocalization was assessed by calculating Pearson’s correlation coefficient with Imaris software. Bar, 13 µm. The results shown are representative of three independent experiments.

Article Snippet: The antibodies used were directed against the FLAG (anti-DDK; Origene) and HA epitopes (Sigma-Aldrich) and were used at a dilution of 1:500.

Techniques: Derivative Assay, Comparison, Quantitative RT-PCR, Control, Retroviral, Transduction, Selection, Transfection, Immunoprecipitation, Incubation, Western Blot, Plasmid Preparation, Immunofluorescence, Imaging, Software

Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: Analysis of zinc signaling/levels and NF- k B activation in the presence and absence of CIB1. (A) HEK293T cells were transfected with plasmids encoding CIB1, EVER1, and EVER2 either alone or in combination and with a 4×MRE-dependent EGFP reporter construct. After 24 h, cells were stimulated overnight with PMA/ionomycin (10 ng/ml and 50 ng/ml, respectively) or zinc sulfate (ZnSO 4 ; 100 µM). The next day, cells were stained with 1 µg/ml DAPI to exclude dead cells, and GFP fluorescence was determined with an LSRII flow cytometer. The RRR with the value for vector-transfected cells was set at 100%. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to the appropriate vector-transfected control (ns, P > 0.05; *, P < 0.05; ***, P < 0.001; n = 3). (B) Flow cytometric quantification of absolute amounts of labile zinc in LCLs derived from healthy controls, EVER1-, EVER2-, or CIB1-deficient patients, or in keratinocytes from P14 with 1 µM FluoZin-3 as described by . Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to the healthy controls ( n = 3). (C) Kinetics of zinc flux in LCLs derived from healthy controls and EVER1-, EVER2-, or CIB1-deficient patients. Cells were loaded with 1 µM FluoZin-3 for 30 min. Fluorimetric measurement were performed on a Victor microplate reader. Baseline fluorescence was recorded every minute for 10 min. Cells were then loaded with 100 µM ZnSO 4 and recorded for 15 min. The specificity of the zinc signal was confirmed by adding the calcium-specific chelator BAPTA before the quenching of the signal with the zinc-specific chelator TPEN and recording for 20 min. No significant effect of genotype was detected in two-way repeat-measures ANOVA ( n = 3). (D) HEK293T cells were transfected with plasmids encoding CIB1-FLAG, FLAG-EVER1, and EVER2 either alone or in combination. Cells were stimulated with 50 ng/ml TNFα 6 h after transfection, incubated overnight, and then harvested and processed for Western blotting. Membranes were probed for the canonical NF-κB component (p)IκBa and the noncanonical NF-κB component p100/p52. Expression of the constructs used for transfection was verified by incubation with a FLAG-specific antibody. GAPDH served as a loading control ( n = 3). (E) Primary keratinocytes from unrelated donors (controls 1 and 2), a healthy family member from kindred A1 carrying the mutation in a heterozygous state (A1.viii.2), and one patient each from kindreds A1 and C were stimulated with 10 ng/ml TNFα for 5, 10, or 20 min and then harvested and processed for Western blotting. Membranes were probed for the canonical NF-κB component (p)IκBa. CIB1 levels were assessed with a polyclonal antibody. GAPDH served as a loading control. vec, empty vector ( n = 3).

Article Snippet: The antibodies used were directed against the FLAG (anti-DDK; Origene) and HA epitopes (Sigma-Aldrich) and were used at a dilution of 1:500.

Techniques: Activation Assay, Transfection, Construct, Staining, Fluorescence, Flow Cytometry, Plasmid Preparation, Comparison, Control, Derivative Assay, Incubation, Western Blot, Expressing, Mutagenesis

Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: PLA and coimmunoprecipitation in HaCaT. (A) HaCaT cells were transfected with plasmids encoding FLAG-HPV5 E1, E2, E6, and E7, FLAG-HPV16 E1, E2, E5, E6, and E7, FLAG-HPV4 E8, FLAG–CRPV E8, and CIB1-HA alone or in combination. The day after transfection, samples were plated on microscopy slides, allowed to adhere, fixed in acetone, permeabilized, and subjected to Duolink PLAs with rabbit-HA– and mouse-FLAG–specific antibodies. Z stacks were acquired with a widefield microscope, and PLA-positive sites (defined as structures >0.35 µM 2 ) were scored with Imaris software for 15–50 cells per condition. These pooled results were obtained in two independent experiments. The orange bars indicate the mean. (B) HaCaT cells were transfected with plasmids encoding CIB1-HA and the FLAG-tagged HPV E ORFs scoring positive in the PLA in A. 1 d after transfection, samples were subjected to immunoprecipitation (IP) with FLAG-specific antibodies. Western blots were performed to detect coimmunoprecipitated HPV5 E1, HPV16 E2, E5, HPV4 E8, and CRPV E8. The immunoprecipitation of CIB1 was confirmed by reincubation with a FLAG-specific antibody. The presence of all proteins was checked by Western blotting analysis on an input sample taken before immunoprecipitation. GAPDH served as a loading control ( n = 3). vec, vector.

Article Snippet: The antibodies used were directed against the FLAG (anti-DDK; Origene) and HA epitopes (Sigma-Aldrich) and were used at a dilution of 1:500.

Techniques: Transfection, Microscopy, Software, Immunoprecipitation, Western Blot, Control, Plasmid Preparation

Journal: Journal of Cell Science

Article Title: Luman is involved in osteoclastogenesis through the regulation of DC-STAMP expression, stability and localization

doi: 10.1242/jcs.176057

Figure Lengend Snippet: Subcellular localization of Luman during osteoclastogenesis. BMMs were infected with a retroviral vector expressing FLAG-tagged Luman. After viral infection, BMMs were cultured with M-CSF for 2 days. Thereafter, infected BMMs were incubated with M-CSF and RANKL for the indicated time periods. Cells were treated with proteasome inhibitor MG132 4 h before fixation, and then were fixed with cold methanol. Immunostaining was performed with antibodies against FLAG and calnexin (CNX), an ER marker. Nuclear counter-staining was conducted with DAPI. At day 2, signals for Luman in the nucleus as well as in the ER were detected (arrows). Scale bar: 20 µm.

Article Snippet: For supershift experiments, samples were treated with an anti-FLAG antibody (Rockland) at 4°C for 1 h before incubation with a biotin-labeled probe.

Techniques: Infection, Retroviral, Plasmid Preparation, Expressing, Cell Culture, Incubation, Immunostaining, Marker, Staining

Journal: Journal of Cell Science

Article Title: Luman is involved in osteoclastogenesis through the regulation of DC-STAMP expression, stability and localization

doi: 10.1242/jcs.176057

Figure Lengend Snippet: Luman mediates the induction of DC-STAMP through CRE-like sequence in the DC-STAMP promoter. (A) Schematic representation of the 0.2-kb promoter region of the murine DC-STAMP gene. AP-1 site (△), NFAT site (□) and CRE-like sequence site (◆) are indicated. TSS, transcription start site. Each CRE-like sequence is indicated. (B) The reporter plasmid of the 0.2-kb murine DC-STAMP promoter fused with the luciferase gene was co-transfected with the pcDNA empty vector (Mock) or Luman N-terminus pcDNA expression vector (Luman-N) into RAW264 cells. Luciferase activity was measured at 24 h after transfection. Data are from three independent experiments. Values indicate mean±s.e.m. † P <0.001. (C) Luciferase reporter assay with a series of deletion mutants of the DC-STAMP promoter reporter plasmids. Reporter plasmids in which the NFAT-binding site or each CRE-like sequence had been deleted from the DC-STAMP promoter region were used. Reporter activities were measured in the same way as described in B. Data are from three independent experiments. Values indicate mean±s.e.m. ** P <0.01; † P <0.001; n.s., not significant. (D) Electrophoretic mobility shift assay. The biotin-labeled probes, including the CRE(2) site of the DC-STAMP promoter, were incubated with nuclear extract derived from FLAG–Luman-N expressing HeLa cells. Note that the binding of FLAG–Luman-N to the CRE(2) site was abolished by a competitor (lane 3), and a supershift after incubation with anti-FLAG antibodies (lane 4) was detected.

Article Snippet: For supershift experiments, samples were treated with an anti-FLAG antibody (Rockland) at 4°C for 1 h before incubation with a biotin-labeled probe.

Techniques: Sequencing, Plasmid Preparation, Luciferase, Transfection, Expressing, Activity Assay, Reporter Assay, Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Incubation, Derivative Assay

Journal: Journal of Cell Science

Article Title: Luman is involved in osteoclastogenesis through the regulation of DC-STAMP expression, stability and localization

doi: 10.1242/jcs.176057

Figure Lengend Snippet: Luman interacts with DC-STAMP and is localized to the Golgi. (A) Co-immunoprecipitation analyses of HeLa cells expressing Luman and DC-STAMP. Cells were co-transfected with expression plasmids for Luman tagged with FLAG at the N-terminus (FLAG–Luman) and DC-STAMP tagged with HA at the C-terminus (DC-STAMP–HA). Cell lysates were immunoprecipitated with antibodies against HA (a-HA), and the immunoprecipitated (IP) samples were subjected to western blotting (WB) with an antibody against FLAG. (B) Subcellular localization of Luman and DC-STAMP that had been expressed in HeLa cells. FLAG–Luman and/or DC-STAMP–HA expression plasmids were transfected into HeLa cells. The transfected cells were immunostained with antibodies against FLAG or HA. Note that co-expression of Luman and DC-STAMP caused the emergence of perinuclear accumulation. Arrows indicate accumulated signals of Luman and DC-STAMP. Scale bars: 10 µm. (C) Double-staining for FLAG and Golgi markers on the cells expressing FLAG–Luman and DC-STAMP–HA. HeLa cells were co-transfected with FLAG–Luman and DC-STAMP–HA expression plasmids. The transfected cells were immunostained with antibodies against FLAG and GM130 (cis-Golgi marker) or against FLAG and TGN46 (trans-Golgi marker). Note that perinuclear accumulation of FLAG–Luman and DC-STAMP–HA overlaps with that of the trans-Golgi marker. Arrows indicate accumulated signals for Luman and DC-STAMP. Scale bars: 10 µm.

Article Snippet: For supershift experiments, samples were treated with an anti-FLAG antibody (Rockland) at 4°C for 1 h before incubation with a biotin-labeled probe.

Techniques: Immunoprecipitation, Expressing, Transfection, Western Blot, Double Staining, Marker

Journal: Journal of Cell Science

Article Title: Luman is involved in osteoclastogenesis through the regulation of DC-STAMP expression, stability and localization

doi: 10.1242/jcs.176057

Figure Lengend Snippet: Luman defines the localization and stabilization of DC-STAMP by interacting with it. (A) Schematic representation of the domain structure of murine DC-STAMP and mapping of the binding region for Luman. Co-immunoprecipitation experiments with various truncated DC-STAMP mutants revealed the binding region in DC-STAMP for Luman. Solid squares with Roman numerals indicate the transmembrane domains of DC-STAMP. Putative binding regions are highlighted in red. The degrees of association between Luman and the DC-STAMP constructs shown are indicated as follows: circle, strong; triangle, weak; cross, none. Numbers represent amino acid residues. (B) Co-immunoprecipitation followed by western blot analysis. HeLa cells were co-transfected with expression plasmids for FLAG–Luman and full-length DC-STAMP [DC-STAMP(full)–HA] or HA-tagged truncated DC-STAMP [DC-STAMP(1-167)–HA]. Cell lysates were immunoprecipitated (IP) with antibodies against HA (a-HA), and the immunoprecipitated proteins were subjected to western blotting with antibodies against FLAG or HA. Note that Luman only interacts with the full-length DC-STAMP. (C) Immunostaining for FLAG–Luman and DC-STAMP–HA. HeLa cells were co-transfected with FLAG–Luman and DC-STAMP(full)–HA or DC-STAMP(1-167)–HA expression plasmids, and immunostained with antibodies against FLAG (green) and HA (red). Upon co-expression of FLAG–Luman and DC-STAMP(full)–HA, both proteins accumulated at the perinuclear region. DC-STAMP(1-167)–HA was scarcely detected in the absence of treatment with MG132. Arrows indicate accumulated signals for Luman and DC-STAMP. Scale bars: 10 µm. (D) Subcellular fractionation analyses of Luman and DC-STAMP. HeLa cells were co-transfected with expression plasmids for FLAG–Luman and DC-STAMP(full)–HA or DC-STAMP(1-167)–HA. The microsome membranes from cell lysates were ultracentrifuged and fractionated in an iodixanol gradient. Each fraction was subjected to western blotting. TGN46, GM130 and calnexin (CNX) were examined as specific intracellular markers for the trans-Golgi, cis-Golgi and ER, respectively. TGN46 is mainly distributed in fractions 2 and 3; GM130 in fractions 4–6; CNX in fractions 7–9.

Article Snippet: For supershift experiments, samples were treated with an anti-FLAG antibody (Rockland) at 4°C for 1 h before incubation with a biotin-labeled probe.

Techniques: Binding Assay, Immunoprecipitation, Construct, Western Blot, Transfection, Expressing, Immunostaining, Fractionation