Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Sound Rhythms Are Encoded by Postinhibitory Rebound Spiking in the Superior Paraolivary Nucleus

doi: 10.1523/JNEUROSCI.2450-11.2011

Figure Lengend Snippet: SPON neurons express HCN1 and HCN2 channels and have large Ih inwardly rectifying (IR) currents. A, HCN1 immunoreactivity in the superior olivary complex of an adult mouse. The low-magnification micrograph illustrates that the lateral superior olive expresses the strongest HCN1-like immunostaining, the SPON displays moderate levels of expression, and the MNTB shows the weakest immunoreactivity. At higher magnification, it is evident that both somata (arrowheads) and dendrites (arrow) of SPON neurons are immunopositive. Asterisks denote landmarks in slice. Scale bars: left, 200 μm; right, 50 μm. B) HCN2 is most strongly expressed in the MNTB, whereas expression in the LSO is weakest. At higher magnification, it is evident that SPON neurons are also immunopositive. Asterisks denote landmarks in slice. Scale bars: left, 200 μm; right, 50 μm. C, Current traces recorded from an SPON neuron induced by hyperpolarization from a holding current of −62 to −122 mV, in −10 mV voltage steps, under control conditions (top) and during pharmacological blockade of Ih with 20 μM ZD7288 (bottom). D, The size of the IR current was measured at steady state (i.e., ~1.35 s after induction) at each hyperpolarizing voltage (n = 42). The IR currents were significantly diminished by ZD7288 (n = 6) in the SPON neurons. ***p < 0.001, Student’s t test. E, Average activation time constants of the IR currents in SPON neurons were obtained by fitting a single-exponential function to the current traces.

Article Snippet: Sections were incubated in 2% normal donkey serum in blocking solution overnight at 4°C with one of the following primary antibodies: polyclonal rabbit α -HCN1 (1:250, lot number AN-10; Alomone Labs), which is directed against amino acid residues 6–24 of the intracellular N terminus of rat HCN1 (GenBank accession number {"type":"entrez-protein","attrs":{"text":"Q9JKB0","term_id":"29840774","term_text":"Q9JKB0"}} Q9JKB0 ); polyclonal rabbit α -HCN2 (1:400, lot number AN-08; Alomone Labs), directed against amino acids 147–161 of the intracellular N terminus of human HCN2 (GenBank accession number {"type":"entrez-protein","attrs":{"text":"Q9UL51","term_id":"108935843","term_text":"Q9UL51"}} Q9UL51 ); or monoclonal mouse α -HCN2 (1:400, clone N71/37; NeuroMab, UC Davis/NIH NeuroMab Facility) directed against amino acids 761–863 of the C terminus of rat HCN2 (GenBank accession number {"type":"entrez-protein","attrs":{"text":"Q9JKA9","term_id":"83303515","term_text":"Q9JKA9"}} Q9JKA9 ) used in combination with a mouse-on-mouse kit (Vector Laboratories).

Techniques: Immunostaining, Expressing, Activation Assay

Journal: Biomolecules

Article Title: The Degradation of Hyaluronan in the Skin

doi: 10.3390/biom12020251

Figure Lengend Snippet: Overview of HA-degrading proteins.

Article Snippet: The following antibodies were used in the study: Anti-ACTB (Actin-β, sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase, WH0002597M1, Merck Life Science UK Ltd.) anti-HYAL1 (bs-1235R, Bioss, Boston, MA, USA), anti-HYAL2 (bs-5888R, Bioss), anti-CEMIP (SAB2105467, Merck Life Science UK Ltd.), anti-TMEM2 (PA5-85901, Thermo Fisher Scientific, Waltham, MA, USA), Streptavidin-FITC (Fluorescein Isothiocyanate Conjugate, S3762, Merck Life Science UK Ltd.), Streptavidin-Alexa Fluor 647 (S21374, Invitrogen), goat anti-rabbit IgG AlexaFluor647 (A-21245, Thermo Fisher Scientific), goat anti-rabbit IgG AlexaFluor555 (ab150078), IgG (10500C Rabbit IgG Isotype Control, Thermo Fisher Scientific), Hoechst 34580 (H21486, Thermo Fisher Scientific).

Techniques: Activity Assay

Journal: Biomolecules

Article Title: The Degradation of Hyaluronan in the Skin

doi: 10.3390/biom12020251

Figure Lengend Snippet: The expression of HYALs, CEMIP and TMEM2 in the human skin and the detection of proteins via Western blotting. ( A ) The relative expression levels of the HYAL1, HYAL2, HYAL3, HYAL4, HYAL5, CEMIP and TMEM2 transcripts were measured using RT-qPCR. The average values from three independent experiments are shown. The error bars represent the SEM. ( B ) Western blot analyses of the human skin lysates (in duplicates) with anti-HYAL1 and anti-GAPDH antibodies and ( C ) anti-HYAL2 and anti β-actin antibodies. GAPDH and ACTB were used as the loading controls. Abbreviations: Hyaluronidase (HYAL), Transmembrane Protein type II (TMEM2), Cell Migration Inducing Hyaluronan Binding Protein (CEMIP), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Actin Beta (ACTB).

Article Snippet: The following antibodies were used in the study: Anti-ACTB (Actin-β, sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase, WH0002597M1, Merck Life Science UK Ltd.) anti-HYAL1 (bs-1235R, Bioss, Boston, MA, USA), anti-HYAL2 (bs-5888R, Bioss), anti-CEMIP (SAB2105467, Merck Life Science UK Ltd.), anti-TMEM2 (PA5-85901, Thermo Fisher Scientific, Waltham, MA, USA), Streptavidin-FITC (Fluorescein Isothiocyanate Conjugate, S3762, Merck Life Science UK Ltd.), Streptavidin-Alexa Fluor 647 (S21374, Invitrogen), goat anti-rabbit IgG AlexaFluor647 (A-21245, Thermo Fisher Scientific), goat anti-rabbit IgG AlexaFluor555 (ab150078), IgG (10500C Rabbit IgG Isotype Control, Thermo Fisher Scientific), Hoechst 34580 (H21486, Thermo Fisher Scientific).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Migration, Binding Assay

Journal: Biomolecules

Article Title: The Degradation of Hyaluronan in the Skin

doi: 10.3390/biom12020251

Figure Lengend Snippet: Localization of HYAL1, HYAL2, CEMIP and TMEM2 in the human skin. ( A , B ) Immunohistochemical and immunohistofluorescent analysis of IgG, ( C , D ) HYAL1, ( E , F ) HYAL2, ( G , H ) CEMIP and ( I , J ) TMEM2. The insets provide enlarged views of the regions in the gray boxes. The nuclei were stained with Höechst (blue). The asterisks indicate the fibroblasts, and the arrows indicate the keratinocytes. The scale bars at the bottom of the figures represent 50 μm. Abbreviations: hyaluronidase (HYAL), Transmembrane Protein type II (TMEM2), Cell Migration Inducing Hyaluronan Binding Protein (CEMIP).

Article Snippet: The following antibodies were used in the study: Anti-ACTB (Actin-β, sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase, WH0002597M1, Merck Life Science UK Ltd.) anti-HYAL1 (bs-1235R, Bioss, Boston, MA, USA), anti-HYAL2 (bs-5888R, Bioss), anti-CEMIP (SAB2105467, Merck Life Science UK Ltd.), anti-TMEM2 (PA5-85901, Thermo Fisher Scientific, Waltham, MA, USA), Streptavidin-FITC (Fluorescein Isothiocyanate Conjugate, S3762, Merck Life Science UK Ltd.), Streptavidin-Alexa Fluor 647 (S21374, Invitrogen), goat anti-rabbit IgG AlexaFluor647 (A-21245, Thermo Fisher Scientific), goat anti-rabbit IgG AlexaFluor555 (ab150078), IgG (10500C Rabbit IgG Isotype Control, Thermo Fisher Scientific), Hoechst 34580 (H21486, Thermo Fisher Scientific).

Techniques: Immunohistochemical staining, Staining, Migration, Binding Assay

Journal: Biomolecules

Article Title: The Degradation of Hyaluronan in the Skin

doi: 10.3390/biom12020251

Figure Lengend Snippet: A suggested metabolic scheme of the degradation of hyaluronan and the localization of HA-degrading proteins in human skin. HMW HA is hydrolyzed by HYALs. This most probably initially occurs on the plasma membrane via HYAL2. The HA is then internalized into the cell via the use of surface HA scavenger receptors, CEMIP or a binding protein (e.g., CD44) for receptor-mediated endocytosis or macropinocytosis. Once internalized, the HA is degraded by HYAL1, HYAL2 and exoglycosidases in the lysosomes into small fragments and subsequently exocytosed. Modified from Csoka et al. (2001) and Racine and Mummert (2012) . According to our data, this mechanism is thought to occur mainly within dermal fibroblasts and basal keratinocytes. The upper keratinocytes appeared to lack the expression of CEMIP. Abbreviations: Hyaluronidase (HYAL), Transmembrane Protein type II (TMEM2), Cell Migration Inducing Hyaluronan Binding Protein (CEMIP), Cluster of differentiation 44 (CD44).

Article Snippet: The following antibodies were used in the study: Anti-ACTB (Actin-β, sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase, WH0002597M1, Merck Life Science UK Ltd.) anti-HYAL1 (bs-1235R, Bioss, Boston, MA, USA), anti-HYAL2 (bs-5888R, Bioss), anti-CEMIP (SAB2105467, Merck Life Science UK Ltd.), anti-TMEM2 (PA5-85901, Thermo Fisher Scientific, Waltham, MA, USA), Streptavidin-FITC (Fluorescein Isothiocyanate Conjugate, S3762, Merck Life Science UK Ltd.), Streptavidin-Alexa Fluor 647 (S21374, Invitrogen), goat anti-rabbit IgG AlexaFluor647 (A-21245, Thermo Fisher Scientific), goat anti-rabbit IgG AlexaFluor555 (ab150078), IgG (10500C Rabbit IgG Isotype Control, Thermo Fisher Scientific), Hoechst 34580 (H21486, Thermo Fisher Scientific).

Techniques: Binding Assay, Modification, Expressing, Migration

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-knockdown in RF/6A cells by siIGFBP-rP1. (A) Reverse transcription-quantitative PCR analysis of IGFBP-rP1 transcript expression in RF/6A cells. GAPDH served as an internal reference for control. Both siIGFBP-rP1 duplex 1 and 2 significantly inhibited the expression IGFBP-rP1 compared with controls ( # P<0.05). Furthermore, the inhibitory effect of siIGFBP-rP1 duplex 2 was significantly increased compared with siIGFBP-rP1 duplex 1 (*P<0.05). (B) IGFBP-rP1 expression was measured by western blotting and normalized to that of β-actin. The inhibitory effect of siIGFBP-rP1 on the IGFBP-rP1 protein expression was consistent with that of the RNA expression. The membranes were stripped off and probed for the proteins. Data are presented as the mean ± standard deviation of three independent experiments with similar results and calculated as the integrated optical density of IGFBP-rP1 relative to the internal reference. # P<0.01 vs. the blank control group. *P<0.05 vs. the siIGFBP-rP1 duplex 1 group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA. Lane M, marker; lane 1, blank control; lane 2, transfection reagent; lane 3, scrambled control siRNA; lane 4, siRNA duplex 1; lane 5, siRNA duplex 2.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot, RNA Expression, Standard Deviation, Binding Assay, Marker, Transfection

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell growth curves of siIGFBP-rP1-transfected cells and untransfected cells cultured in normoxic conditions for 6, 12, 24, 48 and 72 h. The OD values of transfected cells at 12, 24 and 48 h were significantly higher compared with untransfected cells (*P<0.01). Furthermore, the OD value of siIGFBP-rP1 duplex 2-transfected cells was significantly higher compared with siIGFBP-rP1 duplex 1-transfected cells at 24 h ( # P<0.01). No significant differences were observed among the groups at 6 and 72 h. OD values are presented as the mean ± standard deviation of 4 wells/group and experiments were performed in triplicate. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA; OD, optical density.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Standard Deviation, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell growth curves of siIGFBP-rP1-transfected or untransfected cells cultured under normoxic or hypoxic conditions for 6, 12, 24, 48 and 72 h, as detected by MTS colorimetric assays. The OD values of hypoxic groups (CoCl 2 and 1% O 2 ) decreased significantly at 12, 24, 48 and 72 h compared with the control group ( # P<0.01). There was no significant difference between the hypoxic groups (P>0.05). The OD value of the siRNA group were significantly increased at 12, 24 and 48 h compared with controls (**P<0.01). Furthermore, the OD values of the transfected cells cultured in hypoxic conditions (CoCl 2 + siRNA group and 1% O 2 + siRNA group) for 12, 24, 48 and 72 h were significantly lower compared with the siRNA group; additionally, the values were significantly higher compared with the hypoxic groups (*P<0.01). No significant differences were identified between the CoCl 2 + siRNA, 1% O 2 + siRNA and control groups (P>0.05). OD values are presented as the mean ± standard deviation of 4 wells/group and experiments were performed in triplicate. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA; CoCl 2 , cobalt chloride; OD, optical density.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Control, Standard Deviation, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell motility of siIGFBP-rP1-transfected or untransfected cells cultured under normoxic or hypoxic conditions for 24 h is detected by wound and Transwell assays. (A) Representative images showing that RF/6A cells migrated across the wound boundary to the blank area (light microscopy; magnification, ×400) and (B) passed across the filter toward the lower surface (light microscopy; magnification, ×200). Hypoxia significantly promoted cell mobility compared with the controls ( # P<0.01). siIGFBP-rP1 transfection (siIGFBP-rP1 group) further enhanced cell migration, as compared with untransfected cells cultured in hypoxic conditions (hypoxia group; *P<0.01). The transfected cells cultured in hypoxic conditions (hypoxia + siRNA group) exhibited a significantly increased migration ability compared with the transfected cells cultured in normoxic conditions (siIGFBP-rP1 group; **P<0.01). Values are presented as the mean ± standard deviation of 4 samples/group and experiments were performed in triplicate and are quantified as the ratio relative to the control group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Light Microscopy, Migration, Standard Deviation, Control, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-silencing stimulates hypoxia-induced tube formation of RF/6A cells. Representative images (inverted phase contrast microscopy; magnification, ×50) demonstrating that RF/6A cells formed capillary-like tube structures within the Matrigel layer in different mediums. RF/6A cells in hypoxic conditions or siIGFBP-rP1-transfected cells significantly formed completely enclosed capillary-like tubes compared with the controls ( # P<0.01 the hypoxia group vs. the control group; *P<0.01 the siIGFBP-rP1 group vs. the control group). Moreover, siIGFBP-rP1 transfection further promoted tube formation in RF/6A cells in the hypoxia + siIGFBP-rP1 group compared with the siIGFBP-rP1 group (**P<0.01). The values are presented as the mean ± standard deviation of 4 samples/group and experiments were performed in triplicate and are quantified as the ratio relative to the control group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific small interfering RNA.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Microscopy, Transfection, Control, Standard Deviation, Binding Assay, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-silencing upregulates the hypoxia-induced RAF/MEK/ERK signaling pathway activation and VEGF expression. Representative images and quantified data demonstrated that hypoxic stress upregulated B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A cells compared with controls ( # P<0.05). siIGFBP-rP1 transfection significantly promoted hypoxia-induced B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A compared with the hypoxia group ( # P<0.05). IGFBP-rP1 restoration significantly downregulated the expression of B-RAF, p-MEK, p-ERK and VEGF in siIGFBP-rP1-transfected cells, under both normoxic and hypoxic conditions, compared with the siIGFBP-rP1 and hypoxia + siIGFBP-rP1 groups, respectively (*P<0.01). The membranes were stripped off and probed for the proteins. Values are presented as the mean ± SD of 3 independent experiments with similar results and are presented as the integrated optical density of studied proteins relative to GAPDH. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA. Lane 1, control; lane 2, siIGFBP-rP1; lane 3, siIGFBP-rP1 + IGFBP-rP1; lane 4, hypoxia; lane 5, hypoxia + siIGFBP-rP1; lane 6, hypoxia + siIGFBP-rP1 + IGFBP-rP1.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Activation Assay, Expressing, Transfection, Binding Assay, Control