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