Journal: The Journal of comparative neurology

Article Title: Cerebellar premotor output neurons collateralize to innervate the cerebellar cortex

doi: 10.1002/cne.23787

Figure Lengend Snippet: Antibodies Used

Article Snippet: For immunolabeling, sections were blocked in a solution of 10% Normal Goat Serum (Invitrogen) in PB or in the permeabilization solution described above (1hr) and then incubated with one or two of the following primary antibodies (host in parenthesis): (mouse) Synaptic Vesicle Protein 2 (SV2; Developmental Studies Hybridoma Bank Cat# sv2, RRID:AB_528480; for 48 hours at 1:400; concentration determined from serial dilution test); (rabbit) anti-metabotropic Glutamate receptor 2+3 (mGluR 2/3; Abcam; Cat# ab6438, Lot# GR12051, RRID: AB_10307; manufacturer recommended dilution) for 24–48 hours at 1:500; (rabbit) anti-neurogranin for 24 hours at 1:500 (Millipore; Cat#AB5620, Lot# 2441899; RRID: AB_91937; manufacturer recommended dilution) ( ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Immunogen Manufacturer, Catalog/Lot number, RRID, Host Species Concentration Used Synaptic Vesicle Protein 2 Transmembrane glycoprotein of ~95kDa on cytoplasmic side of synaptic vesicles Developmental Studies Hybridoma Bank, University of Iowa, Cat # sv2, RRID: AB_528480, mouse monoclonal 1:400 Anti-metabotropic Glutamate receptor 2+3 Peptide (NGREVVDSTTSSL) corresponding to 13 c-terminus amino acid of mGluR2 and 3 in rat Abcam, Cat# ab6438, Lot# GR12051, RRID: AB_10307, rabbit polyclonal 1:500 Anti-Neurogranin Recombinant rat neurogranin (complete sequence) Millipore, Cat# AB5620, Lot# 2441899, RRID: AB_91937, rabbit polyclonal 1:500 Anti-GFP GFP antibody Abcam, Cat# ab6556, Lot# GR154034-1, RRID: AB_305564, rabbit polyclonal 1:50 Open in a separate window Antibodies Used After three washes in PB, sections were then incubated with secondary antibodies at a 1:400 dilution (90 min).

Techniques: Concentration Assay, Recombinant, Sequencing

Journal: The Journal of comparative neurology

Article Title: Cerebellar premotor output neurons collateralize to innervate the cerebellar cortex

doi: 10.1002/cne.23787

Figure Lengend Snippet: BDA- and virally- labeled boutons are immunopositive for SV2. A. (far left) A schematic diagram illustrating site of BDA injection to the ventrolateral thalamus (VL) is shown; (left) A cerebellar mossy fiber rosette located in the Crus I lobule labeled with BDA after a VL injection was immunopositive for SV2 (middle) as shown in the overlay (right; n=3). Confocal stack thickness was 1.35 μm. B–C. (far left) Schematic diagram of site of BDA injections into the granule cell layer. (See text for the range of injection sites for these experiments.) (left) Terminal boutons labeled with BDA in the red nucleus (RN) after a granule cell layer injection at the base of the Simple lobule near the primary fissure (n=5) expressed SV2 (middle) as shown in the overlay (right). Confocal stack thickness was 1.7 μm. C. (left) BDA-labeled terminal boutons were observed in VL following granule cell layer injection at the base of the Simple lobule near the primary fissure (n=5); SV2 immunostaining (middle) overlayed with labeled terminal (right). Confocal stack thickness was 0.34 μm. D–E. D. (far left) A schematic of AAV1-CAG-Flex-eGFP injection into the red nucleus of Ntsr1-Cre mice is shown. (left) Terminal boutons labeled with GFP were observed in the granule cell layer of the 4/5 lobule following virus injections into the RN of Ntsr1-Cre mice (n=5); these terminals expressed SV2 (middle) as shown in overlay (right). Confocal stack thickness was 1.89 μm. E. (left) GFP-expressing terminal boutons were seen in VL following viral injections to the RN of Ntsr1-Cre mice (n=5); SV2 immunostaining (middle) is overlayed with boutons (right). Confocal stack thickness was 1.62 μm. Scales, A,D = 5 μm; B,C and E = 10 μm.

Article Snippet: For immunolabeling, sections were blocked in a solution of 10% Normal Goat Serum (Invitrogen) in PB or in the permeabilization solution described above (1hr) and then incubated with one or two of the following primary antibodies (host in parenthesis): (mouse) Synaptic Vesicle Protein 2 (SV2; Developmental Studies Hybridoma Bank Cat# sv2, RRID:AB_528480; for 48 hours at 1:400; concentration determined from serial dilution test); (rabbit) anti-metabotropic Glutamate receptor 2+3 (mGluR 2/3; Abcam; Cat# ab6438, Lot# GR12051, RRID: AB_10307; manufacturer recommended dilution) for 24–48 hours at 1:500; (rabbit) anti-neurogranin for 24 hours at 1:500 (Millipore; Cat#AB5620, Lot# 2441899; RRID: AB_91937; manufacturer recommended dilution) ( ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Immunogen Manufacturer, Catalog/Lot number, RRID, Host Species Concentration Used Synaptic Vesicle Protein 2 Transmembrane glycoprotein of ~95kDa on cytoplasmic side of synaptic vesicles Developmental Studies Hybridoma Bank, University of Iowa, Cat # sv2, RRID: AB_528480, mouse monoclonal 1:400 Anti-metabotropic Glutamate receptor 2+3 Peptide (NGREVVDSTTSSL) corresponding to 13 c-terminus amino acid of mGluR2 and 3 in rat Abcam, Cat# ab6438, Lot# GR12051, RRID: AB_10307, rabbit polyclonal 1:500 Anti-Neurogranin Recombinant rat neurogranin (complete sequence) Millipore, Cat# AB5620, Lot# 2441899, RRID: AB_91937, rabbit polyclonal 1:500 Anti-GFP GFP antibody Abcam, Cat# ab6556, Lot# GR154034-1, RRID: AB_305564, rabbit polyclonal 1:50 Open in a separate window Antibodies Used After three washes in PB, sections were then incubated with secondary antibodies at a 1:400 dilution (90 min).

Techniques: Labeling, Injection, Immunostaining, Expressing

Journal: The Journal of comparative neurology

Article Title: Cerebellar premotor output neurons collateralize to innervate the cerebellar cortex

doi: 10.1002/cne.23787

Figure Lengend Snippet: Close contacts between nucleocortical terminals and Golgi cells were observed and express SV2. Each row follows a similar convention, showing terminal label at far left, followed by SV2 immunostaining, then Golgi cell markers and finally an overlay of the three signals. A. (far left) Nucleocortical mossy fiber bouton was labeled with ventrolateral thalamus BDA injection (n=3); (left middle) SV2 immunostaining colocalized with the labeled terminal; (right middle) neurogranin immunostaining was used to identify Golgi cells; (far right) an overlay of the channels illustrated SV2-expressing mossy fibers form close contacts with Golgi cell. Confocal stack thickness was 0.81μm. Arrowheads highlight sites of putative contacts onto Golgi cells. B. Conventions as in A but for mGluR2/3 immunostaining (n=3). Confocal stack thickness was 0.27μm. C. Conventions as in A but for glycinergic GFP-expressing Golgi cell (n=3). Confocal stack thickness was 2.16 μm. D. (far left) Conventions as in A but showing virally labeled nucleocortical mossy fiber. (left middle) (n=5) Confocal stack thickness was 2.16 μm. E. Conventions as in D but for mGluR2/3 immunostaining following red nucleus viral injection (n=3). Confocal stack thickness was 1.62 μm.Scales, 5μm.

Article Snippet: For immunolabeling, sections were blocked in a solution of 10% Normal Goat Serum (Invitrogen) in PB or in the permeabilization solution described above (1hr) and then incubated with one or two of the following primary antibodies (host in parenthesis): (mouse) Synaptic Vesicle Protein 2 (SV2; Developmental Studies Hybridoma Bank Cat# sv2, RRID:AB_528480; for 48 hours at 1:400; concentration determined from serial dilution test); (rabbit) anti-metabotropic Glutamate receptor 2+3 (mGluR 2/3; Abcam; Cat# ab6438, Lot# GR12051, RRID: AB_10307; manufacturer recommended dilution) for 24–48 hours at 1:500; (rabbit) anti-neurogranin for 24 hours at 1:500 (Millipore; Cat#AB5620, Lot# 2441899; RRID: AB_91937; manufacturer recommended dilution) ( ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Immunogen Manufacturer, Catalog/Lot number, RRID, Host Species Concentration Used Synaptic Vesicle Protein 2 Transmembrane glycoprotein of ~95kDa on cytoplasmic side of synaptic vesicles Developmental Studies Hybridoma Bank, University of Iowa, Cat # sv2, RRID: AB_528480, mouse monoclonal 1:400 Anti-metabotropic Glutamate receptor 2+3 Peptide (NGREVVDSTTSSL) corresponding to 13 c-terminus amino acid of mGluR2 and 3 in rat Abcam, Cat# ab6438, Lot# GR12051, RRID: AB_10307, rabbit polyclonal 1:500 Anti-Neurogranin Recombinant rat neurogranin (complete sequence) Millipore, Cat# AB5620, Lot# 2441899, RRID: AB_91937, rabbit polyclonal 1:500 Anti-GFP GFP antibody Abcam, Cat# ab6556, Lot# GR154034-1, RRID: AB_305564, rabbit polyclonal 1:50 Open in a separate window Antibodies Used After three washes in PB, sections were then incubated with secondary antibodies at a 1:400 dilution (90 min).

Techniques: Immunostaining, Labeling, Injection, Expressing

Journal: American Journal of Cancer Research

Article Title: SSH3 facilitates colorectal cancer cell invasion and metastasis by affecting signaling cascades involving LIMK1/Rac1

doi:

Figure Lengend Snippet: Expression of SSH3 in CRC and its correlation with CRC prognosis. A. qRT-PCR analysis of SSH3 expression in 32 paired CRC tissues; SSH3 was quantified relative to the matched adjacent no tumor tissues. Error bars represent means ± SD calculated from three parallel experiments. B. Expression analyses of SSH3 protein in 7 surgical CRC tissues and the paired normal intestine epithelial samples using Western blot. β-tubulin was used as a loading control. C. Immunostaining of SSH3 protein in CRC tissue samples and normal colorectal tissues. D. Statistical analyses of the average SSH3 immunostaining score between normal intestinal tissues and CRC specimens with different degrees of differentiation, TNM classification and Metastasis (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). E. Immunostaining of SSH3 protein in CRC tissue samples diagnosed with tumor thrombus and the paired lymph node metastasis. F. Kaplan-Meier survival analyses published in PROG gene revealed that a higher expression of SSH3 was significantly correlated with poorer survival of patients.

Article Snippet: Immunofluorescence analysis Cells were seeded onto coverslips at a density of 5 × 10 4 per well for 48 h and then probed with primary antibodies against SSH3 (Proteintech Group, Chicago, MA, USA) and LIMK1 (Abcam, USA) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Immunostaining

Journal: American Journal of Cancer Research

Article Title: SSH3 facilitates colorectal cancer cell invasion and metastasis by affecting signaling cascades involving LIMK1/Rac1

doi:

Figure Lengend Snippet: The relationship between the expression of SSH3 and clinicopathological parameters

Article Snippet: Immunofluorescence analysis Cells were seeded onto coverslips at a density of 5 × 10 4 per well for 48 h and then probed with primary antibodies against SSH3 (Proteintech Group, Chicago, MA, USA) and LIMK1 (Abcam, USA) overnight at 4°C.

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: SSH3 facilitates colorectal cancer cell invasion and metastasis by affecting signaling cascades involving LIMK1/Rac1

doi:

Figure Lengend Snippet: Spearman correlation analysis between the expression of SSH3 and Clinicopathological Features

Article Snippet: Immunofluorescence analysis Cells were seeded onto coverslips at a density of 5 × 10 4 per well for 48 h and then probed with primary antibodies against SSH3 (Proteintech Group, Chicago, MA, USA) and LIMK1 (Abcam, USA) overnight at 4°C.

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: SSH3 facilitates colorectal cancer cell invasion and metastasis by affecting signaling cascades involving LIMK1/Rac1

doi:

Figure Lengend Snippet: Up-regulation of SSH3 promoted the invasion and metastasis of CRC cell in vitro. A. Overexpression of SSH3 in SW480 and SW620 cells analyzed through Western blot. β-tubulin was used as a loading control. B. The overexpression of SSH3 lead to a marked increase in the number of protrusions in SW480 and SW620 cells in three-dimensional morphogenesis assays. The bar chart represents the filopodia/cell. Error bars represent the means ± SD calculated from three parallel experiments. C. Overexpression of SSH3 increased cell migration as determined by transwell assays. The bar chart represents the migration cell numbers. Error bars represent the means ± SD of 5 different fields. D. Wound healing assay used to determine the migration ability of cells with up-regulated SSH3.

Article Snippet: Immunofluorescence analysis Cells were seeded onto coverslips at a density of 5 × 10 4 per well for 48 h and then probed with primary antibodies against SSH3 (Proteintech Group, Chicago, MA, USA) and LIMK1 (Abcam, USA) overnight at 4°C.

Techniques: In Vitro, Over Expression, Western Blot, Control, Migration, Wound Healing Assay

Journal: American Journal of Cancer Research

Article Title: SSH3 facilitates colorectal cancer cell invasion and metastasis by affecting signaling cascades involving LIMK1/Rac1

doi:

Figure Lengend Snippet: Down-regulation of SSH3 inhibited the invasion and metastasis of CRC cell in vitro. A. Silencing of SSH3 in shRNA-transduced stable HT29 and HCT116 cells. β-tubulin was used as a loading control. B. Reduction of endogenous SSH3 lead to a marked decrease in the number of protrusions in three-dimensional morphogenesis assays. The bar chart represents the filopodia/cell. Error bars represent the means ± SD calculated from three parallel experiments. C. Down-regulation of SSH3 decreased cell migration as determined by transwell assays. The bar chart represents the migration cell numbers. Error bars represent the means ± SD of 5 different fields. D. Wound healing assay used to determine the migration ability of cells with down-regulated SSH3.

Article Snippet: Immunofluorescence analysis Cells were seeded onto coverslips at a density of 5 × 10 4 per well for 48 h and then probed with primary antibodies against SSH3 (Proteintech Group, Chicago, MA, USA) and LIMK1 (Abcam, USA) overnight at 4°C.

Techniques: In Vitro, shRNA, Control, Migration, Wound Healing Assay

Journal: American Journal of Cancer Research

Article Title: SSH3 facilitates colorectal cancer cell invasion and metastasis by affecting signaling cascades involving LIMK1/Rac1

doi:

Figure Lengend Snippet: SSH3 promotes the metastatic potential of CRC cells in vivo. A and C. The representative gross images of the lungs from different experimental groups are shown. Sections of the liver were stained with H&E. B and D. Box-scatter plots show the number of metastatic nodules in the lungs as observed in each group. E. The representative gross images of the intestines and livers from different experimental groups are shown. Sections of the liver were stained with H&E. F. Box-scatter plot shows the number of metastatic nodules in the liver as observed in each group.

Article Snippet: Immunofluorescence analysis Cells were seeded onto coverslips at a density of 5 × 10 4 per well for 48 h and then probed with primary antibodies against SSH3 (Proteintech Group, Chicago, MA, USA) and LIMK1 (Abcam, USA) overnight at 4°C.

Techniques: In Vivo, Staining

Journal: American Journal of Cancer Research

Article Title: SSH3 facilitates colorectal cancer cell invasion and metastasis by affecting signaling cascades involving LIMK1/Rac1

doi:

Figure Lengend Snippet: High expression of SSH3 promotes the invasion and metastasis of CRC cells by regulating the cytoskeleton. A. Analyses of SSH3-regulated gene signatures via GSEA. B. Co-localization of SSH3 and F-actin in CRC cells. C and D. Rearrangements of cytoskeleton in CRC cells when SSH3 was up- or downregulated.

Article Snippet: Immunofluorescence analysis Cells were seeded onto coverslips at a density of 5 × 10 4 per well for 48 h and then probed with primary antibodies against SSH3 (Proteintech Group, Chicago, MA, USA) and LIMK1 (Abcam, USA) overnight at 4°C.

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: SSH3 facilitates colorectal cancer cell invasion and metastasis by affecting signaling cascades involving LIMK1/Rac1

doi:

Figure Lengend Snippet: SSH3 is involved in the cytoskeleton signaling pathway by interacting with LIMK1 and Rac1. A. Colocalization of SSH3 and LIMK1 in CRC cells. B. Extracts of HT29 cells were subjected to Co-IP using SSH3 antibody or control IgG, and Western blot was performed with LIMK1 and Rac1 antibodies. Reciprocal Co-IPs were performed using LIMK1, Rac1 and IgG antibodies, followed by Western blot with SSH3, Rac1 or LIMK1 antibodies. C. Expression of LIMK1, p-LIMK1, Cofilin and p-Cofilin when SSH3 was up-regulated or down-regulated using Western blot. GAPDH was using as a loading control.

Article Snippet: Immunofluorescence analysis Cells were seeded onto coverslips at a density of 5 × 10 4 per well for 48 h and then probed with primary antibodies against SSH3 (Proteintech Group, Chicago, MA, USA) and LIMK1 (Abcam, USA) overnight at 4°C.

Techniques: Co-Immunoprecipitation Assay, Control, Western Blot, Expressing

Journal: American Journal of Cancer Research

Article Title: SSH3 facilitates colorectal cancer cell invasion and metastasis by affecting signaling cascades involving LIMK1/Rac1

doi:

Figure Lengend Snippet: A model of SSH3 involvement in CRC. SSH3 may promote the invasion and metastasis of CRC by interacting with LIMK1 and Rac1 which can modulate the activity of Cofilin and the rearrangements of cytoskeleton.

Article Snippet: Immunofluorescence analysis Cells were seeded onto coverslips at a density of 5 × 10 4 per well for 48 h and then probed with primary antibodies against SSH3 (Proteintech Group, Chicago, MA, USA) and LIMK1 (Abcam, USA) overnight at 4°C.

Techniques: Activity Assay