Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Secretagogin and other CBPs . PFA-fixed rat brain slices were immunostained for Secretagogin (Scgn: green) and other CBPs (red) and processed for immunofluorescence. (A-C) Hippocampal CA1 region: Secretagogin (green), Parvalbumin (PV, red). (A) Scale bar: 100 μm . (B) Arrows indicate co-expression of Scgn and PV; green arrow: Scgn only, red arrow: PV only; yellow arrow: co-expression of Scgn and PV in a single neuron. Scale bar: 50 μm. (C) Larger magnification of Scgn/PV co-expressing neurons indicates a distinct subcellular distribution. Scale bar: 7.5 μm. (D) Secretagogin-positive neurons near the ventricle. Secretagogin (green), Calbindin D28k (red). Scale bar: 25 μm. (E) Habenula: Secretagogin (green), Calbindin D28k (red). Scale bar: 75 μm. (F) Habenula: Secretagogin (green), Calretinin (red). Scale bar: 100 μm.

Article Snippet: The following commercial antibodies were used for immunofluorescence or other techniques as indicated: rabbit anti-pan-TAU (Cat. No. A 0024, DakoCytomation, Denmark, Glostrup) dil. 1:5000 for immunoblotting; mouse anti-Parvalbumin (Cat. No. PVG214, Swant, Switzerland) dil. 1:3000; mouse anti-Calbindin D28k (Cat. No.300, Swant, Switzerland) dil. 1:10,000; mouse anti-Calretinin (Cat. No. 6B3, Swant, Switzerland) dil. 1:5000; mouse anti-GRP78 (which was a kind gift from the lab of Prof. Johannes Berger) dil. 1:100; mouse anti-GM130 (Cat. No. 560257, BD Biosciences, Franklin Lakes, NJ) dil. 1:100; mouse monoclonal anti-β-actin (AC-15; Cat. No. NB600-501, Novus Biologicals, Littleton, CO, USA) for Western blot controls dil. 1:5000.

Techniques: Immunofluorescence, Expressing

Journal: Human Molecular Genetics

Article Title: Protein kinase C activity is a protective modifier of Purkinje neuron degeneration in cerebellar ataxia

doi: 10.1093/hmg/ddy050

Figure Lengend Snippet: Increased cerebellar PKC substrate phosphorylation is observed in SCA1. (A) Representative western blot for phosphorylated PKC substrates at 5 weeks of age showing increased PKC substrate phosphorylation in cerebella from ATXN1[82Q]tg/tg mice, summarized in (B). Tubulin was used as a loading control. (C) Representative western blot for phosphorylated PKC substrates at 15 weeks of age show increased PKC substrate phosphorylation in cerebella from ATXN1[82Q]tg/tg mice, summarized in (D). Tubulin was used as a loading control. (E) Representative immunohistochemistry experiment showing PKC substrate phosphorylation (green) in Purkinje neurons (Calbindin: red) from the anterior cerebellar vermis in 15-week-old wild-type and ATXN1[82Q]tg/tg mice. Scale bars, 50 µm. (F) Western blot demonstrating increased phosphorylation of Ser880 of GluA2 in ATXN1[82Q]tg/tg mice at 15 weeks of age, summarized in (G). Total GluA2 was used as a loading control. (H) Representative images of phosphorylated PKC substrate staining (green) in calbindin-stained (red) Purkinje neurons from an SCA1 patient sample and healthy control sample. Scale bars, 50 µm. (I) Summarized data show an increase in phosphorylated PKC substrate-to-calbindin staining ratio in the somatic compartment of Purkinje neurons from SCA1 patient samples and age-matched healthy control samples. Each data point represents an individual patient, with error bars representing SEM for measurements from multiple Purkinje neurons in that individual. (J) Phosphorylated PKC substrate staining intensity in the somatic compartment shows a significant increase in SCA1 patient samples relative to age-matched healthy control samples. Each data point represents an individual patient, with error bars representing S.E.M. for measurements from multiple Purkinje neurons in that individual. Throughout, data are represented as means with error bars representing S.E.M. * P<0.05, ** P <0.01; *** P <0.001. Statistical significance derived by unpaired two-tailed Student’s t-test (B, D, G) or two-way ANOVA (I, J).

Article Snippet: Purkinje neurons were labeled with mouse anti-calbindin (1: 1000, Cat. no. C9848, Sigma-Aldrich) and goat anti-mouse Alexa594-conjugated secondary antibody (1: 200, Ref. no. A11005, Life Technologies Invitrogen).

Techniques: Western Blot, Immunohistochemistry, Staining, Derivative Assay, Two Tailed Test

Journal: Human Molecular Genetics

Article Title: Protein kinase C activity is a protective modifier of Purkinje neuron degeneration in cerebellar ataxia

doi: 10.1093/hmg/ddy050

Figure Lengend Snippet: Increased PKC substrate phosphorylation is a protective modifier of neurodegeneration in SCA1 mice. (A) Representative western blot for PKC substrate phosphorylation in wild-type, PKCitg/-, ATXN1[82Q]tg/- and ATXN1[82Q]tg/-; PKCitg/- mice at 20 weeks of age demonstrating reduction of PKC substrate phosphorylation in ATXN1[82Q]tg/-; PKCitg/- mice. Tubulin was used as a loading control. (B) Reduced molecular layer thickness in lobule V of ATXN1[82Q]tg/-; PKCitg/- mice at 20 weeks of age. (C) Whole-cell capacitance measurements in ATXN1[82Q]tg/-; PKCitg/- Purkinje neurons from the anterior cerebellar vermis show a greater reduction in cell size relative to ATXN1[82Q]tg/- Purkinje neurons, which are atrophied relative to wild-type and PKCitg/- Purkinje neurons at 20 weeks of age. (D) Representative images at the cerebellar primary fissure in wild-type, PKCitg/-, ATXN1[82Q]tg/- and ATXN1[82Q]tg/-; PKCitg/- mice stained with calbindin at 20 weeks of age. Scale bar, 100 µm. Throughout, data are represented as means with error bars representing S.E.M. * P<0.05. Statistical significance derived by one-way ANOVA with Holm-Sidak multiple comparison test (B, C).

Article Snippet: Purkinje neurons were labeled with mouse anti-calbindin (1: 1000, Cat. no. C9848, Sigma-Aldrich) and goat anti-mouse Alexa594-conjugated secondary antibody (1: 200, Ref. no. A11005, Life Technologies Invitrogen).

Techniques: Western Blot, Staining, Derivative Assay

Journal: Human Molecular Genetics

Article Title: Protein kinase C activity is a protective modifier of Purkinje neuron degeneration in cerebellar ataxia

doi: 10.1093/hmg/ddy050

Figure Lengend Snippet: Increased PKC substrate phosphorylation is a shared protective pathway in SCA2 mice. (A) Representative western blot for PKC substrate phosphorylation in wild-type and ATXN2[127Q]tg/- cerebella at 12 weeks of age demonstrating increased PKC substrate phosphorylation, summarized in (B). Tubulin was used as a loading control. (C) Expression of Dagla is reduced at 12 weeks in ATXN2[127Q]tg/- cerebella as assessed using quantitative RT-PCR. (D) Representative western blot for PKC substrate phosphorylation in wild-type, PKCitg/-, ATXN2[127Q]tg/- and ATXN2[127Q]tg/-; PKCitg/- mice at 25 weeks of age demonstrating reduction of PKC substrate phosphorylation in ATXN2[127Q]tg/-; PKCitg/- mice. Tubulin was used as a loading control. (E) Representative images at the cerebellar primary fissure in sections from wild-type, PKCitg/-, ATXN2[127Q]tg/- and ATXN2[127Q]tg/-; PKCitg/- mice stained with calbindin at 25 weeks of age. Scale bar, 100 µm. (F) Reduced molecular layer thickness in lobule VI of ATXN2[127Q]tg/-; PKCitg/- mice at 25 weeks of age. Throughout, data are represented as means with error bars representing S.E.M. NS = not significant; * P <0.05; ** P <0.01. Statistical significance derived by unpaired two-tailed Student’s t-test (B, C) or one-way ANOVA with Holm-Sidak multiple comparison test (F).

Article Snippet: Purkinje neurons were labeled with mouse anti-calbindin (1: 1000, Cat. no. C9848, Sigma-Aldrich) and goat anti-mouse Alexa594-conjugated secondary antibody (1: 200, Ref. no. A11005, Life Technologies Invitrogen).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Staining, Derivative Assay, Two Tailed Test

Journal: Journal of Cell Science

Article Title: A polycystin-2 protein with modified channel properties leads to an increased diameter of renal tubules and to renal cysts

doi: 10.1242/jcs.259013

Figure Lengend Snippet: Polycystic kidney disease in Pkd2 poreL1 knock-in mice. (A,B) Transverse kidney sections from 12-month-old wild-type (+/+) and homozygous Pkd2 poreL1 knock-in (p/p) were stained with H&E. Whereas kidney cysts can be easily seen at the age of 12 months on a 129/Sv background, no kidney cysts were observed on a C57Bl/6 background over a period of 12 months. Scale bars: 1 mm. (C,D) Immunohistochemical staining of kidney sections from homozygous Pkd2 poreL1 knock-in mice on a 129/Sv background for aquaporin-2 (a marker of principal cells in collecting ducts) shows that cysts originate in collecting ducts (C). Double-immunofluorescence staining for aquaporin-2 (green) and calbindin (a marker of connecting tubules, red) demonstrates the sudden transition from a calbindin-positive connecting tubule to an aquaporin-2-positive collecting duct cyst (asterisk in D). Scale bars: 100 µm. (E–H) Scanning electron micrographs were taken from kidneys of 3-, 6- and 12-month-old wild-type and Pkd2 poreL1 knock-in mice of both sexes on a 129/Sv background to determine the length of primary cilia. No obvious morphological alterations of primary cilia in collecting ducts were detected (E). Scale bars: 2 µm. At all three ages the length of primary cilia in the knock-in mice is shifted towards higher values. The number of cilia analyzed is given in the upper right-hand corner of the line graphs in panels F–H. Images are representative of at least three animals per condition.

Article Snippet: The following primary antibodies were used: a mouse monoclonal anti-calbindin antibody (Merck, cat. no. C8666; diluted 1:4000), a polyclonal goat anti-aquaporin-2 antibody (Santa Cruz Biotechnology, cat. no. sc-9882; diluted 1:200), a rabbit polyclonal anti-laminin antibody (Merck, cat. no. L9393; diluted 1:100), a polyclonal rabbit anti-Na + /Cl − cotransporter antiserum (diluted 1:1000) , a polyclonal rabbit anti-uromodulin antibody (Biotrend, cat. no. 8595-0004; diluted 1:100), and the polyclonal rabbit anti-polycystin-2 antibodies YCB9 and YCC2 (diluted 1:200) ( ).

Techniques: Knock-In, Staining, Immunohistochemical staining, Marker, Double Immunofluorescence Staining