Journal: Scientific Reports

Article Title: The Na +/ H + -Exchanger NHE1 Regulates Extra- and Intracellular pH and Nimodipine-sensitive [Ca 2+ ] i in the Suprachiasmatic Nucleus

doi: 10.1038/s41598-019-42872-w

Figure Lengend Snippet: NHE1 distribution ( A ) and colocalisation with markers for specific cell types ( B – D ) and major inputs ( E , F , G ). (A) NHE1 immunoreactivity is distributed throughout the rostrocaudal axis of the SCN (encircled by the dotted lines). Scale bar: 200 µm. OC: optic chiasm. 3 V: third ventricle. (B – G1) Low magnification images showing the double staining pattern of NHE1 with neuropeptides NP2 ( B ), GRP ( C ), and VIP ( D ) as well as markers for afferent inputs vGluT2 ( E ), NPY ( F ), and SERT ( G1 ). Scale bar: 100 µm. Insets: High magnification images showing individual cells with double staining. Scale bar: 10 µm. Asterisks mark Hoechst-stained nuclei. (G2 ) High magnification image showing high degree of colocalisation (yellow) between NHE1 (green) and SERT (red). Scale bar: 10 µm.

Article Snippet: For immunofluorescence staining, sections (20 μm) were washed for 20–30 min in PBS and then incubated overnight at 4 °C in PBS containing 2% serum, 0.3% Triton X-100, and primary antibodies against NHE1 (rabbit anti-NHE1; 1:100; ab67314, RRID:AB_1141782; Abcam, Cambridge, MA, USA), neurophysin II (NP2) (goat anti-NP2; 1:500; sc-27093, RRID:AB_2061964; Santa Cruz, CA, USA), vasoactive intestinal peptide (VIP) (guinea pig anti-VIP; 1:500; T-5030, RRID:AB_518690; Peninsula Laboratories, San Carlos, CA, USA), gastrin-releasing peptide (GRP) (goat anti-GRP; 1:100; sc-7788, RRID:AB_2232721; Santa Cruz, CA, USA), vesicular glutamate transporter type 2 (vGluT2) (guinea pig anti-vGluT2; 1:300; AB2251, RRID:AB_1587626; Millipore, Temecula, CA, USA), serotonin transporter (SERT) (mouse anti-SERT; 1:200; MAB1564, RRID:AB_94220; Millipore, Temecula, CA, USA), neuropeptide Y (NPY) (goat anti-NPY; 1:300; NBP1-46535, RRID:AB_10009813; Novus, Littleton, CO, USA), CaV1.2 (guinea pig anti-CaV1.2; 1:100; AGP-001; RRID:AB_11219156; Alomone Labs, Jerusalem, Israel), NCX1 (mouse anti-NCX1, against epitope between amino acid 371 and 525 on intracellular side of plasma membrane; 1:100; AB2869, RRID:AB_2191134; Abcam, MA, USA), and Bassoon (mouse anti-Bassoon; 1:200; ADI-VAM-PS003-D, RRID:AB_2038857; Enzo Life Sciences, Farmingdale, NY, USA).

Techniques: Double Staining, Staining

Journal: Journal of Biological Chemistry

Article Title: Hox Proteins Functionally Cooperate with the GC Box-binding Protein System through Distinct Domains

doi: 10.1074/jbc.m303932200

Figure Lengend Snippet: FIG. 2. The GC box but not the HBS is essential for cooperative stimulation by HoxA-13 and Sp1. A, schematic representations of 260 mouse Bmp-4-reporter construct (260wt), its internal deletion (260dHBS) or mutation (260mut) constructs, and SV40 promoter- reporter construct. In the 260dHBS-reporter construct, the “ATTA” sequence was internally deleted in the Hox binding site. In the 260mut reporter construct nucleotides were mutated from “GG” to “TT” in the GC box, indicated by asterisks. TATA indicates the consen- sus TATA box. B, effect of the internal deletion of Hox binding site in the Bmp-4-reporter construct. Each construct was co-transfected with Sp1 or HoxA-13 alone or both Sp1 and HoxA-13. C, effect of GG to TT substitution in mouse 260 Bmp-4-reporter construct and effect of HoxA-13 on SV40 promoter. Each construct was co-transfected with a HoxA-13 expression vector or pMiw control vector.

Article Snippet: Binding reactions were performed with 30 g of the lysates, which were diluted 1:1 with distilled water, and Cy5.5-labeled oligonucleotides containing a GC box consensus site (5 -ATTCGATCGGGGCGGGGCGAGC-3 ), named SP1BS, or 2 Hox binding site (5 -TCAATTAAATGATCAATTAAATGA-3 ), named NP2, in binding buffer (10 mM Tris-HCl (pH 7.5), 75 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 6% glycerol, 1% Nonidet P-40) on ice for 30 min. For the supershift assay, lysates were preincubated with anti-Sp1 antibody (sc59G; Santa Cruz) or anti-HoxA-13 antibody (see below) on ice for 20 min.

Techniques: Construct, Mutagenesis, Sequencing, Binding Assay, Transfection, Expressing, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: Hox Proteins Functionally Cooperate with the GC Box-binding Protein System through Distinct Domains

doi: 10.1074/jbc.m303932200

Figure Lengend Snippet: FIG. 3. Involvement of the GC box-binding proteins in Bmp-4 promoter transcriptional activation by HoxA-13 or HoxD-13. A, effect of various GC box-binding proteins on HoxA-13-dependent transcriptional stimulation. Cells were co-transfected with 250 ng of 260 mouse Bmp-4-reporter construct and 100 ng of Sp family (Sp1, Sp3, Sp4, TIEG1, or BTEB1) expression vector or pMiw control vector, together with 500 ng of HoxA-13 expression vector (filled bars) or pMiw control vector (open bars). The bars indicate S.D. B, effect of Sp1 on HoxD-13-dependent transcriptional stimulation. Cells were co-transfected with 250 ng of 260 mouse Bmp-4-reporter construct and 100 ng of Sp1 expression vector or pMiw control vector, together with 500 ng of HoxD-13 expression vector (filled bars) or pMiw control vector (open bars). The bars indicate S.D. C, effect of HoxA-13 on Sp1 and other GC box-binding proteins bound to GC box. Nuclear extracts from transfected NG108-15 cells with either pMiw control vector, HoxA-13 or Sp1 alone, or both Sp1 and HoxA-13 were incubated with Cy5.5-labeled probes, containing a GC box (SP1BS; top panel) or 2 Hox binding site (NP2; bottom panel) in the absence or presence of antibodies against Sp1 or HoxA-13. Using SP1BS as probe (top), the shift band derived from Sp1, which supershifted (indicated by S.S.) in the presence of Sp1 antibody and increased in extracts from Sp1-transfected cells, was observed as indicated. One major shift band derived from HoxA-13, which inhibited in the presence of HoxA-13 antibody, was observed when NP2 was used as probe and was incubated with extracts from HoxA-13-transfected cells (bottom).

Article Snippet: Binding reactions were performed with 30 g of the lysates, which were diluted 1:1 with distilled water, and Cy5.5-labeled oligonucleotides containing a GC box consensus site (5 -ATTCGATCGGGGCGGGGCGAGC-3 ), named SP1BS, or 2 Hox binding site (5 -TCAATTAAATGATCAATTAAATGA-3 ), named NP2, in binding buffer (10 mM Tris-HCl (pH 7.5), 75 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 6% glycerol, 1% Nonidet P-40) on ice for 30 min. For the supershift assay, lysates were preincubated with anti-Sp1 antibody (sc59G; Santa Cruz) or anti-HoxA-13 antibody (see below) on ice for 20 min.

Techniques: Binding Assay, Activation Assay, Transfection, Construct, Expressing, Plasmid Preparation, Control, Incubation, Labeling, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: Hox Proteins Functionally Cooperate with the GC Box-binding Protein System through Distinct Domains

doi: 10.1074/jbc.m303932200

Figure Lengend Snippet: FIG. 4. Characterization of functional domains of HoxA-13. A, subcellular localization of wild type HoxA-13 and mutant and fusion proteins was monitored by immunostaining with anti-HoxA13 antibody. Immunostaining was performed 36 h after transfection. Approximately the same level of expression was observed for all proteins. Wild type HoxA-13, A13-dAD, A11N-A13HD, A13N-A11HD, and A13–3A proteins were seen in the nucleus, whereas A13-dHD and A13-N191 proteins were seen in the cytoplasm. Both nuclear and cytoplasmic staining were observed in A13-dC33-transfected cells. B, schematic representation of HoxA-13 mutated and fusion constructs (left) and their effect on mouse and chick Bmp-4-reporter transcription (right). The level of each reporter activity, which was co-transfected with pMiw control vector, was set at 1.0. The WFQ sequence in the wild type HoxA-13 helix 3 was changed to AAA in the A13-3A construct. N-terminal region of HoxA-11 and HoxA-13 homeodomain were fused in A11N-A13HD; the N-terminal region of HoxA-13 was followed by the HoxA-11 homeodomain in the A13N-A11HD construct. The bars indicate S.D. values. C, synergism between Sp1 and the chimeric construct of HoxA-13. Cells were cotransfected with 260 mouse Bmp-4-reporter construct and an expression vector carrying deletion constructs of HoxA-13, together with 100 ng of Sp1 expression vector or pMiw control vector. The bars indicate S.D. values. Note that Sp1 did not enhance the activity of A13-dHD, although it enhanced wild type HoxA-13 and other mutation or fusion constructs. D, effect of co-expression of the HoxA-13 N-terminal domain. Cells were co-transfected with the 260 mouse Bmp-4 reporter construct and an expression vector harboring deletion constructs of HoxA-13, together with 500 ng of A13-N191 expression vector or pMiw control vector. Transactivation levels by each expression construct alone were set at 100% individually. The bars indicate S.D. values.

Article Snippet: Binding reactions were performed with 30 g of the lysates, which were diluted 1:1 with distilled water, and Cy5.5-labeled oligonucleotides containing a GC box consensus site (5 -ATTCGATCGGGGCGGGGCGAGC-3 ), named SP1BS, or 2 Hox binding site (5 -TCAATTAAATGATCAATTAAATGA-3 ), named NP2, in binding buffer (10 mM Tris-HCl (pH 7.5), 75 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 6% glycerol, 1% Nonidet P-40) on ice for 30 min. For the supershift assay, lysates were preincubated with anti-Sp1 antibody (sc59G; Santa Cruz) or anti-HoxA-13 antibody (see below) on ice for 20 min.

Techniques: Functional Assay, Mutagenesis, Immunostaining, Transfection, Expressing, Staining, Construct, Activity Assay, Control, Plasmid Preparation, Sequencing

Journal: Journal of Biological Chemistry

Article Title: Hox Proteins Functionally Cooperate with the GC Box-binding Protein System through Distinct Domains

doi: 10.1074/jbc.m303932200

Figure Lengend Snippet: FIG. 6. Suppression of HoxA-13 and Sp1-dependent transcriptional stimulation by Msx-1 and HoxA-11. A, dosage effects of MSX-1 on transactivation of HoxA-13 and Sp1. 250 ng of 260 mouse Bmp-4-reporter construct and 500 ng of wtHoxA-13 or Sp1 expression vector were co-transfected into cells with 250 or 500 ng of MSX-1 expression vector or 500 ng of pMiw control vector (MSX-1; ). B, dosage effects of HoxA-11 on the transactivation by wild type HoxA-13 and fusion constructs. 250 ng of 260 mouse Bmp-4-reporter construct and 500 ng of the expression vector carrying wild type HoxA-13 or two fusion constructs (A11N-A13HD and A13N-A11HD) were co-transfected into cells along with 250 or 500 ng of HoxA-11 expression vector or 500 ng of pMiw control vector. C, effect of HoxA-11 on Sp1. Cells were transfected with 260 mouse Bmp-4-reporter construct and 500 ng of Sp1 expression vector or pMiw control vector, together with 250 ng of HoxA-11 expression vector or pMiw control vector. The bars indicate S.D. values.

Article Snippet: Binding reactions were performed with 30 g of the lysates, which were diluted 1:1 with distilled water, and Cy5.5-labeled oligonucleotides containing a GC box consensus site (5 -ATTCGATCGGGGCGGGGCGAGC-3 ), named SP1BS, or 2 Hox binding site (5 -TCAATTAAATGATCAATTAAATGA-3 ), named NP2, in binding buffer (10 mM Tris-HCl (pH 7.5), 75 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 6% glycerol, 1% Nonidet P-40) on ice for 30 min. For the supershift assay, lysates were preincubated with anti-Sp1 antibody (sc59G; Santa Cruz) or anti-HoxA-13 antibody (see below) on ice for 20 min.

Techniques: Construct, Expressing, Plasmid Preparation, Transfection, Control

Journal: Journal of Biological Chemistry

Article Title: Hox Proteins Functionally Cooperate with the GC Box-binding Protein System through Distinct Domains

doi: 10.1074/jbc.m303932200

Figure Lengend Snippet: FIG. 8. A multifunctional model for HoxA-13 through its three independent activation domains (AD1–3). A, HoxA-13 contains three independent activation domains (AD1–3). AD1 and AD3 can stimulate the Sp1-dependent transcription system individually. AD2 activates transcription in the cytoplasm independently from the Sp1 transcription system. The function of helix 3 is crucial for both Sp1-dependent and -independent transactivation of HoxA-13. B, comparison of the encoded amino acid sequence of the homeodomain exon of chicken HoxA-13, HoxD-13, HoxA-11, and HoxD-11. The conserved patch of each paralog is indicated by a box. The asterisks indicate identical amino acids among the four sequences.

Article Snippet: Binding reactions were performed with 30 g of the lysates, which were diluted 1:1 with distilled water, and Cy5.5-labeled oligonucleotides containing a GC box consensus site (5 -ATTCGATCGGGGCGGGGCGAGC-3 ), named SP1BS, or 2 Hox binding site (5 -TCAATTAAATGATCAATTAAATGA-3 ), named NP2, in binding buffer (10 mM Tris-HCl (pH 7.5), 75 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 6% glycerol, 1% Nonidet P-40) on ice for 30 min. For the supershift assay, lysates were preincubated with anti-Sp1 antibody (sc59G; Santa Cruz) or anti-HoxA-13 antibody (see below) on ice for 20 min.

Techniques: Activation Assay, Comparison, Sequencing

Journal: Scientific Reports

Article Title: The Na +/ H + -Exchanger NHE1 Regulates Extra- and Intracellular pH and Nimodipine-sensitive [Ca 2+ ] i in the Suprachiasmatic Nucleus

doi: 10.1038/s41598-019-42872-w

Figure Lengend Snippet: NHE1 distribution ( A ) and colocalisation with markers for specific cell types ( B – D ) and major inputs ( E , F , G ). (A) NHE1 immunoreactivity is distributed throughout the rostrocaudal axis of the SCN (encircled by the dotted lines). Scale bar: 200 µm. OC: optic chiasm. 3 V: third ventricle. (B – G1) Low magnification images showing the double staining pattern of NHE1 with neuropeptides NP2 ( B ), GRP ( C ), and VIP ( D ) as well as markers for afferent inputs vGluT2 ( E ), NPY ( F ), and SERT ( G1 ). Scale bar: 100 µm. Insets: High magnification images showing individual cells with double staining. Scale bar: 10 µm. Asterisks mark Hoechst-stained nuclei. (G2 ) High magnification image showing high degree of colocalisation (yellow) between NHE1 (green) and SERT (red). Scale bar: 10 µm.

Article Snippet: For immunofluorescence staining, sections (20 μm) were washed for 20–30 min in PBS and then incubated overnight at 4 °C in PBS containing 2% serum, 0.3% Triton X-100, and primary antibodies against NHE1 (rabbit anti-NHE1; 1:100; ab67314, RRID:AB_1141782; Abcam, Cambridge, MA, USA), neurophysin II (NP2) (goat anti-NP2; 1:500; sc-27093, RRID:AB_2061964; Santa Cruz, CA, USA), vasoactive intestinal peptide (VIP) (guinea pig anti-VIP; 1:500; T-5030, RRID:AB_518690; Peninsula Laboratories, San Carlos, CA, USA), gastrin-releasing peptide (GRP) (goat anti-GRP; 1:100; sc-7788, RRID:AB_2232721; Santa Cruz, CA, USA), vesicular glutamate transporter type 2 (vGluT2) (guinea pig anti-vGluT2; 1:300; AB2251, RRID:AB_1587626; Millipore, Temecula, CA, USA), serotonin transporter (SERT) (mouse anti-SERT; 1:200; MAB1564, RRID:AB_94220; Millipore, Temecula, CA, USA), neuropeptide Y (NPY) (goat anti-NPY; 1:300; NBP1-46535, RRID:AB_10009813; Novus, Littleton, CO, USA), CaV1.2 (guinea pig anti-CaV1.2; 1:100; AGP-001; RRID:AB_11219156; Alomone Labs, Jerusalem, Israel), NCX1 (mouse anti-NCX1, against epitope between amino acid 371 and 525 on intracellular side of plasma membrane; 1:100; AB2869, RRID:AB_2191134; Abcam, MA, USA), and Bassoon (mouse anti-Bassoon; 1:200; ADI-VAM-PS003-D, RRID:AB_2038857; Enzo Life Sciences, Farmingdale, NY, USA).

Techniques: Double Staining, Staining