Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: Effects of oxygen tension on retinal levels of HIF-1α, VEGF and β3-AR from PD7 to PD17. ( A ) Schematic diagram of the OIR model including DMOG administration daily from PD7 to PD12. ( B ) Representative blots showing protein levels of HIF-1α, VEGF and β3-AR as evaluated by Western blot in retinal extracts at different times from normoxic controls or OIR mice without or with DMOG administration. β-actin was used as the loading control. ( C – E ), Relative densitometric analyses of the protein levels of HIF-1α, VEGF and β3-AR. ( F ) Retinal mRNA levels of β3-AR at different times from controls or OIR mice untreated or treated with DMOG. * p < 0.05 vs. normoxic controls. One-way ANOVA followed by Tukey’s multiple comparison post-hoc test. Each histogram represents the mean ± SEM of data from 6 independent samples.

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques: Western Blot, Control, Comparison

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: Schematic representation of mouse and human β3-AR genes including their upstream sequences. ( A ) In the mouse gene, 5 exons (E1–E5; solid boxes) and 4 introns (dashed lines) are depicted. They potentially express up to 6 different alternative mRNAs of which 3 codify for the canonical β3-AR protein (yellow mRNAs) while the other 3 for an alternative β3-AR protein with a different C-terminal sequence (purple mRNAs). The putative transcription-start site (TSS) is indicated by the red arrow. The positions of the 6 potential HBSs relative to the TSS are in green. All of them contain the minimal HBS consensus sequence (underlined sequence 5′-ACGTG-3′). ( B ) In the human gene, the positions of the 6 potential HBSs relative to the TSS are in green. All of them contain the minimal consensus sequence (underlined sequence 5′-ACGT-3′). The putative TSS is indicated by the red arrow. The highly conserved nucleotides G −2 and/or C +5 in the mouse and human HBSs sequence are highlighted in red.

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques: Sequencing

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: HIF-1α modeling and HIF-1/DNA docking. ( A ) Root-mean-square (RMS) displacement of protein backbone (black arrow indicates the time at which the stabilization of the protein structure occurs). ( B ) RMS fluctuation of aminoacid displacement relative to the starting structure and the principal domains of the HIF-1α protein, accordingly colored in ( C ). ( D ) HIF-1α protein modelized in its dimeric form showing the correct interaction with the DNA fragment. The two monomers are reported in green and orange respectively, while the DNA fragment is highlighted in blue. The binding site generated by dimerization is better shown in the focus section.

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques: Binding Assay, Generated

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: Docking analysis of model 1 and model 3. ( A ) HIF-1/HBS #1 best association complex: full structure and focus on HIF-1-DNA interactions (boxes). ( B ) HIF-1/HBS #3 best association complex: full structure and focus on HIF-1-DNA interactions (boxes).

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques:

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: HIF-1α interaction with HBS #1 and corresponding β3-AR gene expression at PD12 (from 0 to 12 h of hypoxia) or at PD17. ( A ) Schematic diagram of the OIR model pointing to the specific times under analysis. ( B ) Data from HIF-1α chromatin immunoprecipitation and HBS #1-specific qPCR (ChIP-qPCR) represented as fold enrichment relative to IgG input. ( C ) Corresponding levels of β3-AR mRNA. White bars represent data from retinas of normoxic controls while grey bars represent data from hypoxic mice. One-way ANOVA followed by Tukey’s multiple comparison post-hoc test. Each histogram represents the mean ± SEM of data from 6 independent samples. * p < 0.05 vs. normoxic controls ( n = 6 samples).

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques: Gene Expression, Chromatin Immunoprecipitation, ChIP-qPCR, Comparison

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Association of the α 2 δ 1 Subunit with Ca v 3.2 Enhances Membrane Expression and Regulates Mechanically Induced ATP Release in MLO-Y4 Osteocytes

doi: 10.1002/jbmr.437

Figure Lengend Snippet: RT-PCR primers used for detection of VSCC subunits and osteocyte markers

Article Snippet: The rabbit anti-β 3 antibody was purchased from Alomone Research Laboratories (Jerusalem, Israel).

Techniques: Sequencing

Journal: Frontiers in Molecular Neuroscience

Article Title: LRRK2 Regulates Voltage-Gated Calcium Channel Function

doi: 10.3389/fnmol.2016.00035

Figure Lengend Snippet: Biophysical properties of HEK293 cells recorded in different experimental conditions .

Article Snippet: The primary antibodies used were: rabbit anti-LRRK2 at 1:500 (MJFF2, c41-2 Abcam), rabbit anti-calcium channel β3 subunit 1:200 (Sigma), rabbit anti-calcium channel α1a subunit 1:200 (Sigma), mouse anti-actin 1:2000, mouse anti-myc 1:1000 (Sigma), rabbit anti-S6 ribosomal protein (S6RP) 1:1000 (Cell Signalling) applied in blocking buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20) and 5% nonfat dry milk, overnight at 4°C.

Techniques: Transfection

Journal: Frontiers in Molecular Neuroscience

Article Title: LRRK2 Regulates Voltage-Gated Calcium Channel Function

doi: 10.3389/fnmol.2016.00035

Figure Lengend Snippet: LRRK2 interacts with the Ca V 2.1 channel. (A) Mouse forebrain specimens obtained from littermate wt and heterozygous BAC hG2019S (GS) mice were cross-linked and then processed for immunoprecipitation with rat anti LRRK2 antibodies. Eluted proteins were resolved by SDS-PAGE and detected with anti LRRK2, anti Ca V 2.1 β3 subunit antibodies, anti Ca V 2.1 α1a and anti S6 ribosomal protein (S6RP). (B,C) The graphs report the yield of protein recovered upon LRRK2 immunoprecipitation expressed as a percentage of relative input (B) and as amount of pulled Ca V 2.1 β3 subunit relative to the amount of pulled LRRK2 (C) . Bars represent mean ± SEM ( n = 4) where * indicates p < 0.05; unpaired t -test.

Article Snippet: The primary antibodies used were: rabbit anti-LRRK2 at 1:500 (MJFF2, c41-2 Abcam), rabbit anti-calcium channel β3 subunit 1:200 (Sigma), rabbit anti-calcium channel α1a subunit 1:200 (Sigma), mouse anti-actin 1:2000, mouse anti-myc 1:1000 (Sigma), rabbit anti-S6 ribosomal protein (S6RP) 1:1000 (Cell Signalling) applied in blocking buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20) and 5% nonfat dry milk, overnight at 4°C.

Techniques: Immunoprecipitation, SDS Page

Journal: PathoGenetics

Article Title: Regulation of TGF-β signalling by Fbxo11 , the gene mutated in the Jeff otitis media mouse mutant

doi: 10.1186/1755-8417-2-5

Figure Lengend Snippet: Immunolocalization in palate tissue . a . Coronal sections through the palate of E15.5 wild-type (WT) and homozygote ( Jf/Jf ) embryos, immunohistochemically stained with antibodies against Fbox11, TGF-β3, TGFβR-I, Smad2, and Smad4. Scale bar 50 μm. Medial edge epithelium (mee), nasal palatal epithelium (ne) and oral palatal epithelium (oe). Graph : comparison of the percentage of epithelial cells positive for Smad2 in E15.5 wild-type and homozygote palates. b . Coronal sections through the palate of E14.5 (before fusion) and E15.5 (after fusion) wild-type (WT) and homozygote ( Jf/Jf ) embryos, immunohistochemically stained with pSmad2 antibody. Scale bar 50 μm. Medial edge epithelium (mee). Graph : comparison of the percentage of epithelial cells positive for nuclear and cytoplasmic localization of pSmad2 in E15.5 wild-type and homozygote palates. c . Coronal sections through the palate of E15.5 wild-type (WT) and homozygote ( Jf/Jf ) embryos, immunohistochemically stained with antibodies against Ki67 and cleaved caspase-3. Scale bar 50 μm. Graph : comparison of the percentage of epithelial cells positive for cleaved caspase-3 in E15.5 wild-type and homozygote palates. P -values were determined using two-tailed T -test.

Article Snippet: Rabbit ABC Staining system (sc-2018 Santa Cruz Biotechnology) and goat ABC staining system (sc-2023 Santa Cruz Biotechnology) were used to develop the specific signals with all the antibodies except for TGF-β3.

Techniques: Staining, Two Tailed Test

Journal: PathoGenetics

Article Title: Regulation of TGF-β signalling by Fbxo11 , the gene mutated in the Jeff otitis media mouse mutant

doi: 10.1186/1755-8417-2-5

Figure Lengend Snippet: Immunolocalization in eyelid tissue . a . Coronal sections through the eye of E16 wild-type (WT) and homozygote ( Jf/Jf ) embryos immunohistochemically stained with antibodies against Fbox11, TGF-β3, TGFβR-I, Smad3, Smad2 and Smad4. Scale bar 200 μm. Graph : comparison of the percentage of epithelial cells positive for Smad2 in E16 upper and lower eyelids. b . Coronal sections through the eyes of E15.5 (before fusion) and E16 (after fusion) in wild-type (WT) and homozygote ( Jf/Jf ) embryos stained with pSmad2 antibody (scale bar 200 μm). Epidermis (ep), basal cells (bc) and dermis (d). Graph : comparison of the percentage of epithelial cells with positive nuclear and cytoplasmic localization of pSmad2 in E16 upper and lower eyelids. c . Coronal sections through the eyes of E16 in wild-type (WT) and homozygote ( Jf/Jf ) embryos stained with Ki67 and cleaved caspase-3 antibodies. Scale bar 200 μm. Graph : comparison of the percentage of epithelial cell positive for Ki67 and cleaved caspase-3 in E16 upper and lower eyelids. P -values were determined using two-tailed T -test comparing each homozygote eyelid with the wild type.

Article Snippet: Rabbit ABC Staining system (sc-2018 Santa Cruz Biotechnology) and goat ABC staining system (sc-2023 Santa Cruz Biotechnology) were used to develop the specific signals with all the antibodies except for TGF-β3.

Techniques: Staining, Two Tailed Test