Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: TRIP8b-deficient mice have altered atrial electrophysiology. ( A ) In vitro electrophysiological measurements from Langendorff-perfused hearts show an increase of atrial refractory period (ARP) and atrioventricular-nodal refractory period (AVNRP) in TRIP8b-deficient mice, without changes in sino-nodal activity (sino-nodal recovery time, SNRT) and heart rate (HR). ( B ) Representative tracings are shown for wild-type and TRIP8b-deficient mice. A, atrial activity; atrium electrophysiological tracings from the atrium; V, ventricular activity; ventricle electrophysiological tracings from the ventricle; black arrowheads mark atrial or ventricular stimulation. ( C ) Ganglionic blockade with 0.5 mM hexamethonium leads to a reduction of AVNRP in TRIP8b-deficient mice. Data are presented as box plots (minimum to maximum, n = 5–11 per genotype) and were compared using an unpaired t -test or Mann–Whitney, as appropriate.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: In Vitro, Activity Assay, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: Trip8b mRNA is present in the cardiac nervous system. ( A ) Exon 6–7 can be amplified in cDNA of ganglia-containing atrial tissue of wild-type but not of TRIP8b-deficient mice (left panel). Quantitative PCR analyses show that exon 8–9, 9–10, and 13–14 of Trip8b are still detectable in knockout mice. Data (normalized to Cdkn1b) are presented as individual data points with SEM ( n = 3, right panel) and were compared via one-way ANOVA followed by Sidaks’ multiple comparison test; ns, not significant. ( B ) Trip8b mRNA can be visualized with RNAscope in situ hybridization in neuronal cell bodies of cardiac ganglia. Black arrows in magnifications point to single neurons with Trip8b mRNAs. ( C ) Trip8b mRNA (black arrows) can be visualized with RNAscope in situ hybridization in cardiac nerves of wild-type mice.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Amplification, Real-time Polymerase Chain Reaction, Knock-Out, In Situ Hybridization

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: Trip8b mRNAs are detectable in the cardiac conduction system to a lower amount than in the intracardiac nervous system. ( A ) Sinus node and ( B ) atrioventricular node (AV node) were identified via hematoxylin and eosin (H&E) staining and Hcn4 RNAscope in situ hybridization. Subsequent sections treated with a probe specific for Trip8b show solitary mRNA spots (black arrows) surrounding the sinus node artery and in the AV node. Nuclei are counterstained with hematoxylin in blue. ( C ) The histogram shows the distribution of Trip8b mRNA spots per cell in the intracardiac nervous system (ICNS, nerves, and ganglia), sinus node, and AV node of wild-type and TRIP8b-deficient mice. Overall, 279–404 cells were analyzed for each region of interest per genotype, n = 2–3 images/genotype. ( D ) Trip8b in situ hybridization (red) detects mRNA in wild-type mice but also, to a lower amount, in knockout mice.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Staining, In Situ Hybridization, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: TRIP8b protein is not detectable in the atrial lysates and the cardiac autonomic nervous system. ( A ) Western blot analysis of brain tissue as positive control detects specific bands for TRIP8b (NBP2-38840, Novusbio) already at 2.5 µg total protein. For the heart, 50 µg atrial or ventricular lysate did not show any specific bands, while HCN4 ( B ) is detectable in both genotypes. ( C ) Immunohistochemistry for TRIP8b (APR-070, Alomone Labs) on paraffin sections was established in the central nervous system, more specifically, the cerebral cortex. Neurons positive for TRIP8b are detectable in the wild-type animals but not cortex of TRIP8b-deficient animals. ( D ) To increase the sensitivity of detection, atrial whole-mount preparations (upper panel shows exemplary staining with αTH ab152, Merck Millipore) were stained and ganglia cut out for confocal microscopy (bottom panel with αTH ab76442, Abcam). No specific signal was obtained for TRIP8b (APR-070, Alomone Labs), and no differences were detectable between the genotypes.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Western Blot, Positive Control, Immunohistochemistry, Staining, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: TRIP8b protein is not detectable in the cardiac conduction system in wild-type mice. Sinus node (upper panel) and atrioventricular node (AV node, bottom panel) were identified by anatomical landmarks and HCN4 staining (green). No staining for TRIP8b (red, APR-070, Alomone Labs) was detectable beyond the background.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: HCN channel expression in intracardiac ganglia. ( A ) In situ hybridization of two exemplary wild-type ganglia for Hcn2 (green) and Hcn4 (red). Both mRNAs are present within the ganglia. Boxed area is magnified in the inlay. ( B ) Gene expression analysis of Hcn2 and Hcn4 in TRIP8b-deficient mice and wild-type littermates. Data are presented as normalized gene expression to Cdkn1b using the formula 2 −ΔCt (box plots, minimum to maximum, n = 6 per genotype) and were compared using Mann–Whitney test.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Expressing, In Situ Hybridization, MANN-WHITNEY

Journal: Autophagy

Article Title: The peroxisomal AAA ATPase complex prevents pexophagy and development of peroxisome biogenesis disorders

doi: 10.1080/15548627.2017.1291470

Figure Lengend Snippet: PEX5 accumulates on the peroxisomal membrane in a ubiquitinated form and signals for pexophagy in cells depleted of peroxisomal AAA-complex components. (A) Immunoblots of the total cell lysates for PEX5 from HeLa cells transfected with plasmids encoding GFP-ubiquitin (GFP-Ub) and RFP-SKL after 2 d of treatment with siRNA as indicated. Cells were also treated with 10 μm chloroquine for 24 h to prevent peroxisome loss. The bracket represents modified PEX5; the arrow indicates PEX5, whereas the asterisks (*, **) indicate nonspecific bands. (B) Graph of the relative densitometry reading of GFP-Ub x -PEX5:PEX5 ratio; where GFP-Ub x -PEX5 represent the higher molecular weight bands indicated in (A). The average (n = 3) ± standard deviation for each condition is shown. Asterisks represent p-values of statistics relative to siCTRL: *p < 0.05. A.U., arbitrary units. (C) Same as (A) but immunoblot for ABCD3. The top dashed arrow represents modified ABCD3; the bottom solid arrow indicates ABCD3. (D) Representative fluorescence images of HeLa cells transfected with siRNA as indicated over 2 consecutive d and immunostained for ABCD3 and PEX5. Scale bars: 20 μm. (E) Graph of the average density of PEX5 punctate structures within a cell (the number of PEX5 puncta per volume of cell [μm ]) of 30 cells per trial (n = 3) ± standard deviation in (D). Asterisks represent p values compared with siCTRL: *p < 0.05. (F) Subcellular fractionation of HeLa cells transfected with GFP-ubiquitin (GFP-Ub) and RFP-SKL plasmids after 2 d of treatment with siRNA and chloroquine as in (A). The post nuclear homozygous lysates (WCL) were fractionated into cytosol and membrane fractions, and immunoblotted with the indicated antibodies. The top dashed arrow represents modified PEX5; the bottom solid arrow indicates PEX5, whereas the asterisk (*) indicates nonspecific bands. (G) Representative fluorescence images of HeLa cells transfected with nontargeting siRNA (siCTRL), siRNA against PEX1 (si PEX1 ), and PEX26 (si PEX26 ), with or without siRNA against PEX5 (si PEX5 ) as indicated and immunostained for ABCD3. Scale bars: 50 μm. (H) Graph of the average ABCD3 density per cell of at least 30 cells per trial (n = 3) ± standard deviation in (G). Asterisks represent p-values: *p < 0.05, **p < 0.01.

Article Snippet: The rabbit polyclonal anti-PEX5 antibody used for immunoblotting (1:1000) was generated by immunizing the New Zealand white rabbit with full-length PEX5–6xHIS (accession #NM_001131023) by standard protocol (Cedarlane, Burlington, Ontario).

Techniques: Membrane, Western Blot, Transfection, Ubiquitin Proteomics, Modification, Molecular Weight, Standard Deviation, Fluorescence, Fractionation

Journal: Autophagy

Article Title: The peroxisomal AAA ATPase complex prevents pexophagy and development of peroxisome biogenesis disorders

doi: 10.1080/15548627.2017.1291470

Figure Lengend Snippet: A model for AAA-dependent pexophagy. The peroxisomal AAA-complex prevents ubiquitin-dependent pexophagy and enables peroxisomal matrix protein import. (A) Cells under basal conditions possess their peroxisomal AAA-complex, consisting of PEX1, PEX6 and PEX26, allowing for the efficient removal of ubiquitinated PEX5. Import of matrix proteins is represented by -PTS1. (B) Defect in any of the AAA-complex genes results in the loss of AAA-complex function and the accumulation of ubiquitinated PEX5. This results in the recruitment of autophagy receptors, NBR1 and SQSTM1, which then results in targeting of peroxisomes to phagophores for degradation via pexophagy. (C) Inhibiting pexophagy with chloroquine (CQ) prevents the degradation of peroxisomes, increasing their half-life, and allowing for the import of matrix proteins. This increase in peroxisome numbers and their subsequent increase in import of peroxisomal matrix enzymes results in improved peroxisomal function.

Article Snippet: The rabbit polyclonal anti-PEX5 antibody used for immunoblotting (1:1000) was generated by immunizing the New Zealand white rabbit with full-length PEX5–6xHIS (accession #NM_001131023) by standard protocol (Cedarlane, Burlington, Ontario).

Techniques: Ubiquitin Proteomics