Journal: Neuroscience

Article Title: Spatio-temporal expression analysis of the calcium-binding protein calumenin in the rodent brain

doi: 10.1016/j.neuroscience.2011.11.069

Figure Lengend Snippet: Schematic representation of mRNA and protein domains in calumenin, and in situ hybridization of calumenin transcripts in the developing embryo mouse brain and postnatal mouse brain. (A) Schematic representation of the arrangement of mRNA and protein domains in calumenin. N: signal peptide; EF: Ca 2+ -binding domain; C: ER retention signal HDEF. (B) Representative in situ hybridization micrographs of X-ray films showing head sagittal sections from embryonic stages (embryonic day (E) 12, E14, E16, and E20). The negative control represents the parallel experiment done using sense RNA as a probe on E12 (upper panel) or E16 mouse embryo (lower panel). Negative controls for E14 and E20 embryos were similar (not shown). The highest signal intensity of calumenin mRNA was detected in germinal zones around ventricles (arrows). Significant expression levels were also observed in the cortex, cerebellum, and the brain stem. (C) Representative micrographs of X-ray films showing sagittal sections of the whole mouse brain at postnatal days 7 (P7) and P14 and of the adult mouse brain. The specific signal for calumenin mRNA was strongest in the cerebellum, the cortex, the thalamus, the hippocampus, and the olfactory bulb of both P7 and P14 mice (arrows). The intensity of the specific signal decreases with age (adult), as compared with the negative control (sense RNA probe; the signal of unspecific hybridization was similar for P7 and P14 mice). Key: 4V, fourth ventricle; Aq, aqueduct; BO, olfactory bulb; Cb, cerebellum; Cx, cortex; HC, hippocampus; LV, lateral ventricle; MB, midbrain; MO, medulla oblongata; Ps, pons; Th, thalamus. Scale bars: 1 mm. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

Article Snippet: To test whether rabbit anti-calumenin antibody (Cat. Nr HPA006018, Atlas Antibodies, Stockholm, Sweden) specifically recognizes calumenin, an antibody-competition experiment was performed.

Techniques: In Situ Hybridization, Binding Assay, Negative Control, Expressing, Hybridization

Journal: Neuroscience

Article Title: Spatio-temporal expression analysis of the calcium-binding protein calumenin in the rodent brain

doi: 10.1016/j.neuroscience.2011.11.069

Figure Lengend Snippet: Distribution of calumenin in different tissues of newborn (P0) and adult mice. (A) Western blots showing specificity of the anti-calumenin antibody. In the upper panel, antibodies preadsorbed by extracts of mock-transfected HEK 293 cells were used for immunostaining of extracts of mock-transfected HEK 293 cells, calumenin-transfected HEK 293 cells, as well as P0 brain membranes. In the lower panels immunostaining for calumenin was lost or strongly reduced in all lanes when the anti-calumenin antibody was preadsorbed by extracts of calumenin-transfected HEK 293 cells. (B) Western blots showing calumenin levels in different organs in P0 vs. adult animals. The same amount of protein was applied to each lane, and the signal of each band was normalized to the sum of intensities of bands in all lanes. Calumenin expression in brain, heart, kidney, lung, and stomach of P0 mice was significantly higher than in adult mice (values are given as mean±standard error of mean (SEM): brain P0 vs. adult: 8.3±0.8 vs. 3.6±1.1; heart P0 vs. adult: 13.0±1.1 vs. 4.7±0.5; kidney P0 vs. adult: 21.0±1.5 vs. 6.6±0.9; lung P0 vs. adult: 12.8±1.0 vs. 9.5±1.0; stomach P0 vs. adult: 14.3±1.6 vs. 6.5±1.2). The experiment was independently performed three times, each time using a different P0 and adult animal pair. In addition, three Western blots were done for each animal pair. Asterisk (*) represents statistically significant difference; * P <0.05, ** P <0.01. a.u., arbitrary units; Ab, antibody; WB, Western blot.

Article Snippet: To test whether rabbit anti-calumenin antibody (Cat. Nr HPA006018, Atlas Antibodies, Stockholm, Sweden) specifically recognizes calumenin, an antibody-competition experiment was performed.

Techniques: Western Blot, Transfection, Immunostaining, Expressing

Journal: Neuroscience

Article Title: Spatio-temporal expression analysis of the calcium-binding protein calumenin in the rodent brain

doi: 10.1016/j.neuroscience.2011.11.069

Figure Lengend Snippet: Calumenin expression in different brain regions during postnatal development. Representative blots depicting changes of expression levels of calumenin in different brain regions in the first three postnatal weeks. Equal amounts of protein from three independent experiments were blotted to the membrane, and the signal of each band was normalized to the sum of intensities of bands in all lanes. Calumenin levels in cerebellum, cortex, and hippocampus, tend to decrease in the third postnatal week (Cb: P0, 11.8±2.9; P7, 12.0±2.4; P14, 11.1±0.9; P21, 7.9±1.3; Cx: P0, 8.7±0.6; P7, 10.5±1.6; P14, 8.5±0.6; P21, 5.7±0.5; Hip: P0, 7.9±1.7; P7, 9.9±2.2; P14, 5.9±0.6; P21, 2.9±1.1). The experiment was independently performed three times. For each experiment different animals were used, one for each time point. Three Western blots were done per each experiment. Asterisk represents statistically significant differences (* P <0.05). Key: Cb, cerebellum; Cx, cortex; Hip, hippocampus; n.s., non-significant.

Article Snippet: To test whether rabbit anti-calumenin antibody (Cat. Nr HPA006018, Atlas Antibodies, Stockholm, Sweden) specifically recognizes calumenin, an antibody-competition experiment was performed.

Techniques: Expressing, Membrane, Western Blot

Journal: Neuroscience

Article Title: Spatio-temporal expression analysis of the calcium-binding protein calumenin in the rodent brain

doi: 10.1016/j.neuroscience.2011.11.069

Figure Lengend Snippet: Tissue distribution of calumenin protein in brain sections of newborn (P0) and adult mice. (A) Coronal section of the hippocampal dentate gyrus stained with anti-calumenin antibody pre-adsorbed by extracts of calumenin-transfected HEK 293 cells, showing no signal. (B) Coronal section of the hippocampal region stained with anti-calumenin antibody pre-adsorbed by extracts of mock-transfected HEK 293 cells, showing specific signal in individual cells of subgranular zone and granule cells layer of the dentate gyrus (arrows). (C) Whole brain sagittal section of a P0 mouse brain stained with anti-calumenin antibody. Calumenin displays widespread moderate protein expression with higher signal intensity in cerebellum (Cb), cortex (Cx), olfactory bulb (BO), and migrating cells (MC) around lateral ventricle. (D) Confocal image of the region around the lateral ventricle of P0 mouse brain. High immunoreactivity of calumenin is detected in migrating cells and radial-like glia around the lateral ventricle (arrows). (E) Whole brain sagittal section of an adult mouse brain stained with anti-calumenin antibody. Calumenin protein is moderately expressed throughout the brain. The strongest signal intensity is detected in cerebellum (Cb), olfactory tract (TBO), and thalamic nuclei (Th). (F) Stereomicroscopic image of calumenin immunoreactivity in adult mouse cerebellum. The highest expression of calumenin is visible in Purkinje cell layer (arrowhead) and deep nuclei of cerebellum (arrow). (G) Confocal image of a sagittal adult mouse cerebellar section co-stained for calumenin (green) and a nuclear marker DAPI (blue). The immunoreactivity of calumenin is strongest in PCL (arrow). Some of calumenin-positive cells of PCL send their extensions throughout the molecular cell layer, which is a morphological characteristic of Bergman glia cells (red arrowhead). (H) Confocal image of coronal cerebellar cortex section stained for calumenin (green) and NeuN (red), a postmitotic neuronal marker. The cells in PCL having the strongest signal intensity for calumenin are NeuN-negative (arrow). There are also individual NeuN-negative cells in ML (asterisk) and GCL (crossed arrow) that are moderately positive for calumenin. The arrowhead indicates a large body of a Purkinje cell. (I) Enlarged image of the boxed region from (H)—a group of cells strongly expressing calumenin (arrow) next to the large calumenin-negative cell body of Purkinje cell (arrowhead). (J) Confocal image of cerebellar cortex; calumenin (green), calbindin D28k (red). Calumenin is expressed by cells in PCL (arrow) that send their extension through ML (red arrowhead), which is typical for Bergmann glia. Purkinje cells express calbindin D28k, but no calumenin. (K) Olfactory bulb. A sagittal section was stained for calumenin (green) and nuclear marker DAPI (blue). Individual cells in external plexiform layer are moderately positive for calumenin (arrowhead). (L) Coronal section of pons. Calumenin (green) and NeuN (red) colocalize in individual large neuronal cell bodies (arrow), but there are also NeuN-negative small cells expressing calumenin (arrowhead). (M) Coronal section of the dentate gyrus co-stained for calumenin (green) and NeuN (red). In the basal part of granule cell layer (DGL) there are both NeuN-positive and negative cells of diverse morphology strongly stained for calumenin (arrow). In molecular (DML) and polymorphic layer (PoDG) however, there are only small, individual NeuN-negative cells expressing calumenin (arrowheads). (N) Co-staining of dentate gyrus for calumenin (green) and glial fibrillary acidic protein (GFAP), a marker of astrocytes and neuronal progenitor cells (red). Arrows point to calumenin-positive cells in subgranular zone that co-express GFAP, whereas arrowheads point to astrocytes in DGL and DML, also expressing calumenin. (O) Co-staining of dentate gyrus for calumenin (green) and PSA-NCAM, early neuronal marker (red). Some of calumenin-positive cells in subgranular zone also express PSA-NCAM (arrows). (P) Image of a dentante gyrus section stained for calumenin (green) and NeuN, a postmitotic neuronal marker (red). Arrow points at a large cell in subgranular layer expressing both calumenin and NeuN (weak expression). One cell expressing both proteins is depicted in boxed region at the border between DGL and DML. (Q) Enlarged image of the cell in the boxed region from (P). Arrow points to a weak NeuN signal in the nucleus, whereas arrowhead represents the somatic calumenin staining. Key: BO, olfactory bulb; Cb, cerebellum; Cx, cortex; DG, dentate gyrus; DGL, granule cell layer of dentate gyrus; DML, molecular layer of dentate gyrus; GR, granule cell layer of olfactory bulb; EP, external plexiform layer; GCL, granule cell layer of cerebellum; HC, hippocampus; LV, lateral ventricule; MB, mitral cell body layer; MC, migratory cells; ML, molecular layer of cerebellum; Ps, pons; PCL, Purkinje cell layer; TBO, olfactory tract; Th, thalamus; PoDG, polymorphic layer of dentate gyrus. Scale bars: (A, B) 50 μm, (C) 1 mm, (D) 0.5 mm, (E, F) 1 mm, and (G–P) 50 μm.

Article Snippet: To test whether rabbit anti-calumenin antibody (Cat. Nr HPA006018, Atlas Antibodies, Stockholm, Sweden) specifically recognizes calumenin, an antibody-competition experiment was performed.

Techniques: Staining, Transfection, Expressing, Marker

Journal: Neuroscience

Article Title: Spatio-temporal expression analysis of the calcium-binding protein calumenin in the rodent brain

doi: 10.1016/j.neuroscience.2011.11.069

Figure Lengend Snippet: Calumenin distribution in whole brain cell extracts upon differential centrifugation. (A) Calumenin is abundant in the plasma membrane fraction (P2) and supernatant S1, containing remaining membranes and the cytoplasm, respectively. Further separation reveals relatively high enrichment of calumenin in fraction P3, representing vesicular membranes. In contrast, the soluble fraction S3 contains little calumenin. Detection of actin was used as a loading control, and PSD-95, which is known to be enriched at postsynaptic sites (P2) and at intracellular transport vesicles, is shown as a positive control. (B) The amounts of calumenin per fraction were quantitatively evaluated from three independent experiments (S1, 0.31±0.18; P2, 0.29±0.13; S2, 0.14±0.02; P3, 0.58±0.22; S3, 0.09±0.02). Data were normalized against the respective actin signal, representing the relative amount of protein per lane. a.u., arbitrary units.

Article Snippet: To test whether rabbit anti-calumenin antibody (Cat. Nr HPA006018, Atlas Antibodies, Stockholm, Sweden) specifically recognizes calumenin, an antibody-competition experiment was performed.

Techniques: Centrifugation, Clinical Proteomics, Membrane, Control, Positive Control

Journal: Neuroscience

Article Title: Spatio-temporal expression analysis of the calcium-binding protein calumenin in the rodent brain

doi: 10.1016/j.neuroscience.2011.11.069

Figure Lengend Snippet: Calumenin localization at the subcellular level in primary hippocampal culture. (A) Expression of calumenin during development in hippocampal cell culture from 18 h after plating to DIV 11. The same amount of protein was applied to each lane. Calumenin levels tend to decrease over time in vitro . (B) Hippocampal cell culture showing no signal upon staining with anti-calumenin antibody pre-adsorbed by extracts of calumenin-transfected HEK 293 cells (left panel) and positive staining of cells with calumenin antibody pre-adsorbed with extracts of mock-transfected HEK 293 cells (right panel, arrows). (C–E) Cultured hippocampal cells were co-stained for calumenin (green) and individual markers for different subcellular compartments (red). Calumenin mostly localizes to the somatic region, but it is also present in the neuronal extensions to a much lower extent. (C) Calumenin highly co-localizes to ER marker KDEL (arrows in enlarged boxed region). (D) Calumenin partially co-localizes to Golgi marker GM 130 (arrows in enlarged boxed region). (E) Presynaptic marker synaptophysin does not co-localize to calumenin. Scale bars: (B) 50 μm and (C, D, E) 15 μm.

Article Snippet: To test whether rabbit anti-calumenin antibody (Cat. Nr HPA006018, Atlas Antibodies, Stockholm, Sweden) specifically recognizes calumenin, an antibody-competition experiment was performed.

Techniques: Expressing, Cell Culture, In Vitro, Staining, Transfection, Marker