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: iScience

Article Title: Combined metabolomic and transcriptomic profiling approaches reveal the cardiac response to high-fat diet

doi: 10.1016/j.isci.2022.104184

Figure Lengend Snippet:

Article Snippet: Plasmid: pCMV Flag ERR1 , Addgene , #10975.

Techniques: Marker, Extraction, Transfection, Lysis, Luciferase, Protease Inhibitor, RNA Sequencing, Recombinant, Plasmid Preparation, Software

Journal: International Journal of Molecular Sciences

Article Title: P38 Regulates Kainic Acid-Induced Seizure and Neuronal Firing via Kv4.2 Phosphorylation

doi: 10.3390/ijms21165921

Figure Lengend Snippet: p38 mitogen-activated protein kinase (MAPK) contributed to kainic acid-induced seizure in WT mice but not Kv4.2TA mice. ( A ) Time course of mean behavioral seizure score following kainic acid injection. The mean behavioral seizure score was significantly reduced in Kv4.2TA mice compared to WT mice. Furthermore, p38 inhibitor SB 203580 significantly reduced behavioral seizure score following kainic acid injection in WT mice but not in Kv4.2TA mice, n = 13–15 for each group, two-way ANOVA, * p < 0.05. ( B ) Total behavioral seizure score for each group, n = 13–15 for each group, t -test, * p < 0.05. ( C ) Latency to stage 3 seizure for each group. n = 13–15 for each group, t -test, ** p < 0.01.

Article Snippet: The p38 construct was from Addgene (Watertown, MA, USA, 20351).

Techniques: Injection

Journal: International Journal of Molecular Sciences

Article Title: P38 Regulates Kainic Acid-Induced Seizure and Neuronal Firing via Kv4.2 Phosphorylation

doi: 10.3390/ijms21165921

Figure Lengend Snippet: p38 MAPK contributes to kainic acid-induced Kv4.2 phosphorylation at T607. ( A ) SB 203580, a potent p38 inhibitor (20 mg/kg, i.p., 15 min), blocked kainic acid-induced phosphorylation of Kv4.2 T607 in mouse hippocampus. ( B ) Statistical analysis of the effect of SB 203580 on kainic acid-induced phosphorylation of Kv4.2 at Thr607 in mouse hippocampus, n = 4–6 in each group, t -test, ** p < 0.01.

Article Snippet: The p38 construct was from Addgene (Watertown, MA, USA, 20351).

Techniques: Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: P38 Regulates Kainic Acid-Induced Seizure and Neuronal Firing via Kv4.2 Phosphorylation

doi: 10.3390/ijms21165921

Figure Lengend Snippet: p38 MAPK colocalizes with Kv4.2. ( A ) HEK293T cells were transfected with GFP-Kv4.2 and Flag-p38. Cells were fixed and stained with GFP and Flag to show co-localization. Scale bar: 20 μm. ( B ) High magnification images and line scan analysis of colocalization. Scale bar: 5 μm. ( C ) Mouse brains were co-stained with Kv4.2 and pp38 antibody. Phosphorylated p38 is localized in the cell body and dendrites as well. Scale bar: 20 μm. ( D ) High magnification images showing Kv4.2 and pp38 colocalized in dendrites, as indicated with arrow heads. Scale bar: 5 μm.

Article Snippet: The p38 construct was from Addgene (Watertown, MA, USA, 20351).

Techniques: Transfection, Staining

Journal: International Journal of Molecular Sciences

Article Title: P38 Regulates Kainic Acid-Induced Seizure and Neuronal Firing via Kv4.2 Phosphorylation

doi: 10.3390/ijms21165921

Figure Lengend Snippet: Kainic acid activates p38 MAPK in both WT and Kv4.2TA mice. ( A ) Immunostaining analysis showed p38 phosphorylation increased with kainic acid administration (25 mg/kg, i.p., 30 min) in mouse hippocampus, n = 26 cells in each group, t -test, *** p < 0.001. ( B ) Western blot analysis showed p38 phosphorylation increased with kainic acid administration (25 mg/kg, i.p., 30 min) in hippocampus in both WT and Kv4.2TA mice, n = 4–6 cells in each group, t -test, *** p < 0.001.

Article Snippet: The p38 construct was from Addgene (Watertown, MA, USA, 20351).

Techniques: Immunostaining, Phospho-proteomics, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: P38 Regulates Kainic Acid-Induced Seizure and Neuronal Firing via Kv4.2 Phosphorylation

doi: 10.3390/ijms21165921

Figure Lengend Snippet: p38 impacts hippocampal pyramidal neuron excitability through Kv4.2. ( A ) Current step of +300 pA induces repetitive firing in pyramidal neurons recorded from WT and Kv4.2TA mice with or without SB 203580 treatment. Scale 40 mV/250 ms. Square current inset 300 pA. ( B ) Sequential somatic current injections increasing in magnitude reveal p38 kinase inhibition reduces AP firing frequency in WT hippocampal neurons at +300 pA relative to vehicle ( n = 15 in vehicle, n = 19 in treatment; two-way ANOVA, * p < 0.05). Kv4.2TA neurons display reduced firing frequency at +300 pA relative to WT in vehicle, which is augmented in the presence of SB 203580 such that current magnitudes of +200 and +250 pA also exhibit significant differences ( n = 18 in vehicle, n = 14 in SB 203580; two-way ANOVA, * p < 0.05; *** p < 0.001). ( C ) Inter-spike intervals measured between the first two spikes in a train evoked by 150 pA injection display no significant difference among groups. Kruskal–Wallis test, p > 0.05. ( D ) Ramp current injections evoke repetitive firing in all pyramidal neurons recorded in each condition. Arrow indicates point at which action potential (AP) threshold, rheobase, and latency to fire were measured. Ramp current inset 400 pA/s. ( E ) Minimum current to elicit AP firing at threshold (rheobase) is not significantly different among the populations. One-way ANOVA, p > 0.05. ( F ) Latency to fire in response to ramp injection is not significantly different among populations. Kruskal–Wallis test, p > 0.05.

Article Snippet: The p38 construct was from Addgene (Watertown, MA, USA, 20351).

Techniques: Inhibition, Injection