Journal: Cell reports

Article Title: Chloroquine-Inducible Par-4 Secretion Is Essential for Tumor Cell Apoptosis and Inhibition of Metastasis

doi: 10.1016/j.celrep.2016.12.051

Figure Lengend Snippet: (A) CQ induced Rab8b protein and mRNA levels in a p53-dependent manner. Wild-type (p53 +/+ ) or p53 −/− MEFs were treated with CQ (25 µM) or vehicle (V) for 24 hr; and either the lysates were examined by western blot analysis with the indicated antibodies (left panel), or mRNA prepared from the cells was examined by real-time qRT-PCR (right panel). *p < 0.001, by Student’s t test. (B) p53 directly bound to its consensus-binding site in the Rab8b promoter. MEF cells were treated with CQ or vehicle (V) and subjected to ChIP analysis with p53 antibody (p53 Ab) or control IgG antibody, and immunoprecipitated DNA fragments were amplified and analyzed on agarose gels (left panel) or by real-time qPCR (right panel) with primers near the p53-binding site in Rab8b promoter. The immunoprecipitated fragments were similarly analyzed with random primers for GAPDH promoter or two different primer sets for the Par-4 gene, which does not contain a p53-binding site. *p < 0.001, by Student’s t test. (C) Par-4 co-localized with Rab8b+ vesicles in CQ-treated cells. MEFs were treated with vehicle (V) or CQ (25 µM) in the absence or presence of BFA (1 µg/mL) for 24 hr and subjected to ICC analysis for Par-4 (red fluorescence) and Rab8b (red fluorescence). Cells were stained with DAPI to reveal their nuclei (blue fluorescence). Co-localization of Par-4 and Rab8b vesicles in the overlay images is indicated by yellow fluorescence. Note the dissociation of Par-4 and Rab8b (loss of yellow fluorescence but retention of red and green fluorescence) in the CQ + BFA panel. Cells showing co-localization of Rab8b and Par-4 were scored, and the data are expressed as percentage of cells showing co-localization (right panel). Error bars indicate mean of at least three independent experiments ± SD. See also .

Article Snippet: Human GFP-tagged Rab8B (RG204651) and mouse GFP-tagged Rab8b (MG202204) were from Origene Technologies.

Techniques: Western Blot, Quantitative RT-PCR, Binding Assay, Immunoprecipitation, Amplification, Fluorescence, Staining

Journal: Cell reports

Article Title: Chloroquine-Inducible Par-4 Secretion Is Essential for Tumor Cell Apoptosis and Inhibition of Metastasis

doi: 10.1016/j.celrep.2016.12.051

Figure Lengend Snippet: (A) CQ induced Par-4 secretion by a Rab8b-dependent mechanism. Rab8 wild-type (WT), Rab8b −/− , or Rab8a −/− MEFs were treated with CQ (25 µM) or vehicle (V) for 24 hr, and the CM or lysates were examined by western blot analysis with the indicated antibodies. (B) Induction of Par-4 secretion in response to CQ in Rab8b null cells was restored by re-introduction of Rab8b. Rab8b null MEFs were transiently transfected with GFP-mouse Rab8b (GFP-mRab8b) expression construct or GFP-expression construct for control, and the transfectants were treated with CQ (25 µM) or vehicle for 24 hr. The CM and lysates from the cells were examined by western blot analysis with the indicated antibodies. (C) Par-4 secretion in response to CQ was inhibited by Rab8b siRNAs. Wild-type MEFs were transfected with siRNA duplexes from two different sources, Dharmacon (D) and Santa Cruz Biotechnology (SC), or with scrambled siRNA duplexes for control, and the transfectants were treated with CQ (25 µM) or vehicle for 24 hr. The CM and lysates from the cells were examined by western blot analysis with the indicated antibodies. (D) Introduction of human Rab8b in Rab8b-knockdown MEFs resulted in restoration of Par-4 secretion in response to CQ. MEFs were transfected with the indicated siRNAs, 24 hr later, they were re-transfected with GFP-human Rab8b (GFP-hRab8b) or GFP expression construct, and the transfectants were treated for 24 hr with CQ (25 µM) or vehicle. Western blot analysis of the CM and lysates was performed by using the indicated antibodies. Knockdown of endogenous Rab8b was confirmed with the Rab8b antibody, and expression of GFP-hRab8b was detected with the GFP antibody. See also .

Article Snippet: Human GFP-tagged Rab8B (RG204651) and mouse GFP-tagged Rab8b (MG202204) were from Origene Technologies.

Techniques: Western Blot, Transfection, Expressing, Construct

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mannan-Binding Lectin via Interaction With Cell Surface Calreticulin Promotes Senescence of Activated Hepatic Stellate Cells to Limit Liver Fibrosis Progression

doi: 10.1016/j.jcmgh.2022.03.011

Figure Lengend Snippet: MBL–CRT interaction up-regulates the association of CRT and LRP1 and downstream senescent-related signaling pathways in HSC. LX-2 cells were treated with CRT antibody or IgG 1 hour before the incubation of MBL and H 2 O 2 . ( A ) Representative photomicrographs of the SA–β-gal staining of LX-2 cells. ( B ) The distribution of cell cycle on the treated LX-2 cells with flow cytometry analysis. ( C ) The mRNA levels of senescence-related genes were assessed by quantitative reverse-transcription (qRT)-PCR analysis. ( D ) The association of CRT and LRP1 was determined by immunoblotting of immunoprecipitates in membrane fractions of LX-2 cells. ( E ) The association of LRP1, Rab8, and p110 was determined by immunoblotting of immunoprecipitates in cytoplasm fractions of LX-2 cells. ( F ) The p53, p21, mTOR, p-mTOR, AKT, p-AKT, and p110 protein levels were assessed by Western blot analysis. ( G ) The mRNA levels of fibrosis-associated genes were assessed by qRT-PCR analysis. Scale bars : 100 μm. Data are presented as means ± SEM. ∗∗ P < .01, Student t test or 1-way analysis of variance followed by Tukey post hoc tests for multiple group comparisons. The data shown represent 3 independent experiments. ATPase, adenosine triphosphatase; CCL, chemokine (C-C motif) ligand; CXCL, chemokine (C-X-C motif) ligand; HPF, high-power field; IL, interleukin; MMP, matrix metalloproteinase.

Article Snippet: PI3K antibody (21739-1-AP), Rab8 antibody (55296-1-AP), p53 antibody (60283-2-Ig), p21 antibody (10355-1-AP), p16 antibody (10883-1-AP), Bax antibody (50599-2-Ig), and Bcl2 antibody (12789-1-AP) were supplied by Proteintech (Chicago, IL).

Techniques: Protein-Protein interactions, Incubation, Staining, Flow Cytometry, Reverse Transcription, Quantitative RT-PCR, Western Blot, Membrane

Journal: Journal of Biological Chemistry

Article Title: Rab8b and Its Interacting Partner TRIP8b Are Involved in Regulated Secretion in AtT20 Cells

doi: 10.1074/jbc.m010798200

Figure Lengend Snippet: FIG. 2. In vitro interaction of Rab8b with TRIP8b. A, TRIP8b was in vitro translated in the presence of [35S]methi- onine and incubated with bacterially ex- pressed GST-Rab8b, GST, GST-SLL, or GST-Rac2 bound to glutathione-Sepha- rose beads (as described under “Experi- mental Procedures”). The amount of bound [35S]TRIP8b was determined by autoradiography, quantitated using a densitometer and the values represented as a bar graph. B, TRIP8b binds to GST- Rab8b expressed in mammalian cells in the presence of GTPgS and GDP but with significantly reduced binding in the pres- ence of 10 mM EDTA. TRIP8b also bound Rab8b mutants DSLL and C204S that were expressed in mammalian cells but bound to the two mutants with lesser af- finity compared with GST-Rab8b.

Article Snippet: The amount of immunoprecipitated TRIP8b was determined using an anti-HA monoclonal antibody (F-7, Santa Cruz Biotechnology) and the amount of Rab8b that co-immunoprecipitated with TRIP8b was observed with an anti-Myc monoclonal antibody (9E10, Santa Cruz Biotechnology).

Techniques: In Vitro, Incubation, Autoradiography, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Rab8b and Its Interacting Partner TRIP8b Are Involved in Regulated Secretion in AtT20 Cells

doi: 10.1074/jbc.m010798200

Figure Lengend Snippet: FIG. 3. Co-immunoprecipitation of Rab8b and TRIP8b. HEK 293T cells were co-transfected with HA-TRIP8b and Myc-Rab8b. Cell extracts (lane 1) were incubated with rabbit IgG (lane 2) or anti-HA polyclonal antibodies (lane 3). The immunoprecipitates were electro- phoresed onto a 12% SDS-PAGE gel, transferred to nitrocellulose, and subjected to Western immunoblot analysis. The upper (A) and lower (B) blots were probed with monoclonal antibodies against HA or Myc.

Article Snippet: The amount of immunoprecipitated TRIP8b was determined using an anti-HA monoclonal antibody (F-7, Santa Cruz Biotechnology) and the amount of Rab8b that co-immunoprecipitated with TRIP8b was observed with an anti-Myc monoclonal antibody (9E10, Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Transfection, Incubation, SDS Page, Western Blot, Bioprocessing

Journal: Journal of Biological Chemistry

Article Title: Rab8b and Its Interacting Partner TRIP8b Are Involved in Regulated Secretion in AtT20 Cells

doi: 10.1074/jbc.m010798200

Figure Lengend Snippet: FIG. 7. Co-immunoprecipitation of Rab8b and TRIP8b in the Rab8b stable cells. Postnuclear extracts of AtT20 cells stably express- ing Rab8b were immunoprecipitated with either rabbit IgG or anti- TRIP8b rabbit polyclonal antibody as described under “Experimental Procedures.” Precipitated proteins were subjected to electrophoresis on a 12% SDS-PAGE gel and then probed with antibodies against TRIP8b and anti-Myc monoclonal antibody to detect the co-precipitation of Rab8b.

Article Snippet: The amount of immunoprecipitated TRIP8b was determined using an anti-HA monoclonal antibody (F-7, Santa Cruz Biotechnology) and the amount of Rab8b that co-immunoprecipitated with TRIP8b was observed with an anti-Myc monoclonal antibody (9E10, Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Stable Transfection, Electrophoresis, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Rab8b and Its Interacting Partner TRIP8b Are Involved in Regulated Secretion in AtT20 Cells

doi: 10.1074/jbc.m010798200

Figure Lengend Snippet: FIG. 6. Immunofluorescence localization of Rab8b, TRIP8b, and ACTH in AtT20 stable cells. A, co-localization of Rab8b and ACTH in the AtT20 cells stably expressing Myc-Rab8b or Myc-TRIP8b. Pictures on the left, cells stained with anti-Myc mouse monoclonal antibody and fluorescein isothiocyanate-conjugated anti-mouse anti- body; middle, cells stained with anti-ACTH rabbit polyclonal antibody and rhodamine-conjugated anti-rabbit antibody; right, merge of the left and middle pictures. Bar, 10 mM. B, co-localization of Rab8b and TRIP8b in the Rab8b stable cells. Picture on the left, cells stained with anti- TRIP8b rabbit polyclonal antibody and fluorescein isothiocyanate-con- jugated anti-rabbit antibody; middle, cells stained with anti-Myc mouse monoclonal antibody and rhodamine-conjugated anti-mouse antibody; right, the merge of the former two pictures.

Article Snippet: The amount of immunoprecipitated TRIP8b was determined using an anti-HA monoclonal antibody (F-7, Santa Cruz Biotechnology) and the amount of Rab8b that co-immunoprecipitated with TRIP8b was observed with an anti-Myc monoclonal antibody (9E10, Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Stable Transfection, Expressing, Staining

Journal: Cellular Microbiology

Article Title: Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella ‐containing vacuole

doi: 10.1111/cmi.13151

Figure Lengend Snippet: Hits from shRNA lentiviral screens

Article Snippet: After 48 hr, cells were lysed using ×4 SDS Laemmli sample buffer (Bio‐Rad) and boiled for 5 min. After fractionation on SDS‐polyacrylamide gels (Bio‐Rad), proteins were transferred to nitrocellulose membranes, blocked in 4% (vol/vol) milk in TBST 0.05 M Tris‐buffered saline (NaCl = 0.138 M, KCl = 0.0027 M); (Tween‐20 = 0.05%, pH 8.0) (Sigma‐Aldrich) and probed with antibodies to Rab5A (Cell Signalling, 1:500), Rab5B (Santa Cruz Biotechnology, 1:500), Rab5C (Novus Biologicals, 1:500), Rab11A (Cell Signalling, 1:500), Rab11B (Cell Signalling, 1:500), Rab8A (Cell Signalling Technologies, 1:500), Rab8B (Proteintech, 1:500), EEA1 (BD Biosciences, 1:500), Rab11FIP1 (Cell Signalling, 1:500), VAMP3 (Synaptic Systems, 1:500) or GAPDH (Santa Cruz Biotechnology, 1:1000) in 4% milk/TBST.

Techniques: shRNA

Journal: Cellular Microbiology

Article Title: Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella ‐containing vacuole

doi: 10.1111/cmi.13151

Figure Lengend Snippet: Depletion of anterograde transport Rab8b and a downstream effector reduces bacterial cytosolic exposure. (a) Depletion of Rab8b rescues ΔsdhA vacuole integrity. A/J bone marrow‐derived macrophages were siRNAs treated and knockdown efficiency was assessed (left panels). Nucleofected macrophages were challenged with either WT or ΔsdhA Legionella and percent of cytosol‐detected bacteria was quantified (Experimental Procedures). (b) Growth of noted L pneumophila strains in nucleofected BMDM for 14 hr and quantified as described (Figure ; Experimental Procedures; [Luo & Isberg, ]). (c) Depletion of Rab8 and Rab11 effector VAMP3 rescues ΔsdhA vacuole integrity and growth defects. Nucleofection, vacuole integrity and bacterial yields determined as in panels (A and B). Statistical analyses were performed on normalised data by unpaired t test (*<.05; **<.01; ***<.001; Experimental Procedures)

Article Snippet: After 48 hr, cells were lysed using ×4 SDS Laemmli sample buffer (Bio‐Rad) and boiled for 5 min. After fractionation on SDS‐polyacrylamide gels (Bio‐Rad), proteins were transferred to nitrocellulose membranes, blocked in 4% (vol/vol) milk in TBST 0.05 M Tris‐buffered saline (NaCl = 0.138 M, KCl = 0.0027 M); (Tween‐20 = 0.05%, pH 8.0) (Sigma‐Aldrich) and probed with antibodies to Rab5A (Cell Signalling, 1:500), Rab5B (Santa Cruz Biotechnology, 1:500), Rab5C (Novus Biologicals, 1:500), Rab11A (Cell Signalling, 1:500), Rab11B (Cell Signalling, 1:500), Rab8A (Cell Signalling Technologies, 1:500), Rab8B (Proteintech, 1:500), EEA1 (BD Biosciences, 1:500), Rab11FIP1 (Cell Signalling, 1:500), VAMP3 (Synaptic Systems, 1:500) or GAPDH (Santa Cruz Biotechnology, 1:1000) in 4% milk/TBST.

Techniques: Derivative Assay, Knockdown, Bacteria

Journal: Cell reports

Article Title: Chloroquine-Inducible Par-4 Secretion Is Essential for Tumor Cell Apoptosis and Inhibition of Metastasis

doi: 10.1016/j.celrep.2016.12.051

Figure Lengend Snippet: (A) CQ induced Rab8b protein and mRNA levels in a p53-dependent manner. Wild-type (p53 +/+ ) or p53 −/− MEFs were treated with CQ (25 µM) or vehicle (V) for 24 hr; and either the lysates were examined by western blot analysis with the indicated antibodies (left panel), or mRNA prepared from the cells was examined by real-time qRT-PCR (right panel). *p < 0.001, by Student’s t test. (B) p53 directly bound to its consensus-binding site in the Rab8b promoter. MEF cells were treated with CQ or vehicle (V) and subjected to ChIP analysis with p53 antibody (p53 Ab) or control IgG antibody, and immunoprecipitated DNA fragments were amplified and analyzed on agarose gels (left panel) or by real-time qPCR (right panel) with primers near the p53-binding site in Rab8b promoter. The immunoprecipitated fragments were similarly analyzed with random primers for GAPDH promoter or two different primer sets for the Par-4 gene, which does not contain a p53-binding site. *p < 0.001, by Student’s t test. (C) Par-4 co-localized with Rab8b+ vesicles in CQ-treated cells. MEFs were treated with vehicle (V) or CQ (25 µM) in the absence or presence of BFA (1 µg/mL) for 24 hr and subjected to ICC analysis for Par-4 (red fluorescence) and Rab8b (red fluorescence). Cells were stained with DAPI to reveal their nuclei (blue fluorescence). Co-localization of Par-4 and Rab8b vesicles in the overlay images is indicated by yellow fluorescence. Note the dissociation of Par-4 and Rab8b (loss of yellow fluorescence but retention of red and green fluorescence) in the CQ + BFA panel. Cells showing co-localization of Rab8b and Par-4 were scored, and the data are expressed as percentage of cells showing co-localization (right panel). Error bars indicate mean of at least three independent experiments ± SD. See also .

Article Snippet: Rab8b siRNA duplexes were from Dharmacon (D), and pools of Rab8b siRNA and scrambled siRNA duplexes were from SantaCruz Biotechnology (SC).

Techniques: Western Blot, Quantitative RT-PCR, Binding Assay, Control, Immunoprecipitation, Amplification, Fluorescence, Staining

Journal: Cell reports

Article Title: Chloroquine-Inducible Par-4 Secretion Is Essential for Tumor Cell Apoptosis and Inhibition of Metastasis

doi: 10.1016/j.celrep.2016.12.051

Figure Lengend Snippet: (A) CQ induced Par-4 secretion by a Rab8b-dependent mechanism. Rab8 wild-type (WT), Rab8b −/− , or Rab8a −/− MEFs were treated with CQ (25 µM) or vehicle (V) for 24 hr, and the CM or lysates were examined by western blot analysis with the indicated antibodies. (B) Induction of Par-4 secretion in response to CQ in Rab8b null cells was restored by re-introduction of Rab8b. Rab8b null MEFs were transiently transfected with GFP-mouse Rab8b (GFP-mRab8b) expression construct or GFP-expression construct for control, and the transfectants were treated with CQ (25 µM) or vehicle for 24 hr. The CM and lysates from the cells were examined by western blot analysis with the indicated antibodies. (C) Par-4 secretion in response to CQ was inhibited by Rab8b siRNAs. Wild-type MEFs were transfected with siRNA duplexes from two different sources, Dharmacon (D) and Santa Cruz Biotechnology (SC), or with scrambled siRNA duplexes for control, and the transfectants were treated with CQ (25 µM) or vehicle for 24 hr. The CM and lysates from the cells were examined by western blot analysis with the indicated antibodies. (D) Introduction of human Rab8b in Rab8b-knockdown MEFs resulted in restoration of Par-4 secretion in response to CQ. MEFs were transfected with the indicated siRNAs, 24 hr later, they were re-transfected with GFP-human Rab8b (GFP-hRab8b) or GFP expression construct, and the transfectants were treated for 24 hr with CQ (25 µM) or vehicle. Western blot analysis of the CM and lysates was performed by using the indicated antibodies. Knockdown of endogenous Rab8b was confirmed with the Rab8b antibody, and expression of GFP-hRab8b was detected with the GFP antibody. See also .

Article Snippet: Rab8b siRNA duplexes were from Dharmacon (D), and pools of Rab8b siRNA and scrambled siRNA duplexes were from SantaCruz Biotechnology (SC).

Techniques: Western Blot, Transfection, Expressing, Construct, Control, Knockdown

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