Journal: Molecular Vision

Article Title: GABA Aα1 and GABA Aρ1 subunits are expressed in cultured human RPE cells and GABA A receptor agents modify the intracellular calcium concentration

doi:

Figure Lengend Snippet: Sample ethidium bromide gel of real-time PCR products of gamma-aminobutyric acid Aα1 (GABA Aα1 ) and GABA Aρ1 in cultured human RPE cells (representative image, n = 5). β–Actin (302 bp), GABA Aα1 (211 bp); GABA Aρ1 (263 bp). Abbreviations: NC, negative control; PC, human retina (positive control).

Article Snippet: After being blocked in 5% fat-free milk diluted in Tris-buffered saline with Tween (TBST; 0.1% Tween-20, 150 mM NaCl, 50 mM Tris, pH 7.5) for 1 h, the membrane was incubated with the polyclonal goat anti-human GABA Aα1 antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal goat anti-human GABA Aρ1 antibody (1:100, Santa Cruz, CA), or monoclonal mouse anti-human β-Actin antibody (Zhongshan Jinqiao Company, Beijing, China) overnight at 4 °C, and then was followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000) for 1 h at 37 °C.

Techniques: Real-time Polymerase Chain Reaction, Cell Culture, Negative Control, Positive Control

Journal: Molecular Vision

Article Title: GABA Aα1 and GABA Aρ1 subunits are expressed in cultured human RPE cells and GABA A receptor agents modify the intracellular calcium concentration

doi:

Figure Lengend Snippet: Gamma-aminobutyric acid Aα1 (GABA Aα1 ) and GABA Aρ1 protein expression in cultured human RPE cells detected by western blots (A) and immunofluorescence (B) (representative image; n = 5). A : Specific bands presented at the approximate location of 51 kDa (GABA Aα1 ), 48 kDa (GABA Aρ1 ), and 43 kDa (β-Actin) in lysates of RPE and the retina (positive control, PC). B : Immunofluorescence staining of GABA Aα1 and GABA Aρ1 in cultured human RPE. Nuclei were stained by 4',6-diamidino-2-phenylindole (DAPI; blue; bar = 20 μm). NC, negative control.

Article Snippet: After being blocked in 5% fat-free milk diluted in Tris-buffered saline with Tween (TBST; 0.1% Tween-20, 150 mM NaCl, 50 mM Tris, pH 7.5) for 1 h, the membrane was incubated with the polyclonal goat anti-human GABA Aα1 antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal goat anti-human GABA Aρ1 antibody (1:100, Santa Cruz, CA), or monoclonal mouse anti-human β-Actin antibody (Zhongshan Jinqiao Company, Beijing, China) overnight at 4 °C, and then was followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000) for 1 h at 37 °C.

Techniques: Expressing, Cell Culture, Western Blot, Immunofluorescence, Positive Control, Staining, Negative Control

Journal: Molecular Vision

Article Title: GABA Aα1 and GABA Aρ1 subunits are expressed in cultured human RPE cells and GABA A receptor agents modify the intracellular calcium concentration

doi:

Figure Lengend Snippet: Evidence that the gamma-aminobutyric acid A receptor (GABA A R) agonist muscimol, the antagonist picrotoxin and the GABA Aρ antagonist 1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) can modify the intracellular calcium concentration ([Ca 2+ ]i) in the cultured human RPE. A : Muscimol (100 μM) increased the [Ca 2+ ]i in cultured human RPE cells in buffer with Ca 2+ (paired t-test, p<0.05). B : Pretreatment with picrotoxin (300 μM) completely inhibited the muscimol (100 μM) induced increase of [Ca 2+ ]i in buffer with Ca 2+ . C : Pretreatment with TPMPA (500 μM) completely blocked the muscimol (100 μM) induced increase of [Ca 2+ ]i in buffer with Ca 2+ . D : Muscimol (100 μM) increased the [Ca 2+ ]i in cultured human RPE cells in buffer without Ca 2+ (paired t-test, p<0.05). E : Pretreatment with picrotoxin (300 μM) completely inhibited the muscimol (100 μM) induced increase of [Ca 2+ ]i in buffer without Ca 2+ . F : Pretreatment with TPMPA (500 μM) completely blocked the muscimol (100 μM) induced increase of [Ca 2+ ]i in buffer without Ca 2+ . Left, representative images (1, 8, and 15 images were acquired, respectively). Right, fluorescent intensity in the images acquired before and after muscimol application. The red bar represents the time when muscimol was added. FI represents the fluorescence intensity color scale, with the direction of the arrow indicating higher intensity. *indicates a p<0.05, and ** indicates a p<0.01, compared to the baseline, paired t-test, n=5 for each group.

Article Snippet: After being blocked in 5% fat-free milk diluted in Tris-buffered saline with Tween (TBST; 0.1% Tween-20, 150 mM NaCl, 50 mM Tris, pH 7.5) for 1 h, the membrane was incubated with the polyclonal goat anti-human GABA Aα1 antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal goat anti-human GABA Aρ1 antibody (1:100, Santa Cruz, CA), or monoclonal mouse anti-human β-Actin antibody (Zhongshan Jinqiao Company, Beijing, China) overnight at 4 °C, and then was followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000) for 1 h at 37 °C.

Techniques: Concentration Assay, Cell Culture, Fluorescence

Journal: bioRxiv

Article Title: Neurochondrin interacts with the SMN protein suggesting a novel mechanism for Spinal Muscular Atrophy pathology

doi: 10.1101/183640

Figure Lengend Snippet: mCherry-SmN exhibits similar behaviour to both YFP-SmN and SmB in SH-SY5Y cells. A) SH-SY5Y cells transiently expressing mCherry-SmN and fixed after 24, 48 and 72 hours show variations in distribution of the mCherry-SmN with time. Immunostaining with Y12 (green on overlay) and anti-coilin (white on overlay) shows splicing speckles (arrowheads) and Cajal Bodies (CBs, triangles) respectively. Images are deconvolved z-stacks with 0.2 μm spacing. Bar=7μm. B) mCherry-SmN initially localises diffusely in the cytoplasm, before localising to speckles at the 48 and 72 hour time-points. 3 independent experiments, n=100 cells per experiment. Data shown is mean ± SD.

Article Snippet: Antibodies used were rat monoclonal anti-RFP (Chromotek 5F8, 1∶500); goat polyclonal anti-γCOP (Santa Cruz sc-14167, 1∶250), rabbit polyclonal anti-GFP (Abcam ab290, 1:2000), rabbit polyclonal anti-SNRPN (SmN) (Proteintech 11070-1-AP, 1:800), mouse monoclonal Y12 anti-Smith (SmB) (Abcam ab3138, 1:100), rabbit polyclonal anti-SMN (Santa Cruz sc-15320, 1:500), mouse monoclonal anti-SMN (BD Transduction labs 610646, 1:500), rabbit polyclonal anti-COPB1 (CUSAB CSB-PA005783LA01HU, 1:500), mouse monoclonal anti-Lamin A/C (Santa Cruz sc-7292, 1:500), rabbit polyclonal anti-NCDN (Proteintech 13187-1-AP, 1:500), mouse Monoclonal anti-tubulin (Sigma Aldrich, 1:500) and rabbit polyclonal anti-Histone H3 (Proteintech 17168-1-AP, 1:300).

Techniques: Expressing, Immunostaining

Journal: bioRxiv

Article Title: Neurochondrin interacts with the SMN protein suggesting a novel mechanism for Spinal Muscular Atrophy pathology

doi: 10.1101/183640

Figure Lengend Snippet: A) Immunoblot analysis confirms efficient affinity purification of YFP-SmN, YFP-SmB and YFP. 10% of the affinity purified material (left hand lane in each panel) was compared to 80μg of precleared lysate (Input) and unbound material using anti-GFP. GFP-Trap effectively immunoprecipitated all three proteins. B) After processing the mass spectrometry data, and sorting identified proteins into groups based on Gene Ontology annotations, the interactomes of SmN and SmB are very similar. C) Neurochondrin (NCDN) was identified in the interactome of YFP-SmN, with 5 unique peptide hits encompassing 9% sequence coverage. Each Ion score (Mascot Ion Score) was above the threshold for peptide identity (Mascot Identity Score), with 2 out of the 5 identified peptides having a score of above double the threshold score.

Article Snippet: Antibodies used were rat monoclonal anti-RFP (Chromotek 5F8, 1∶500); goat polyclonal anti-γCOP (Santa Cruz sc-14167, 1∶250), rabbit polyclonal anti-GFP (Abcam ab290, 1:2000), rabbit polyclonal anti-SNRPN (SmN) (Proteintech 11070-1-AP, 1:800), mouse monoclonal Y12 anti-Smith (SmB) (Abcam ab3138, 1:100), rabbit polyclonal anti-SMN (Santa Cruz sc-15320, 1:500), mouse monoclonal anti-SMN (BD Transduction labs 610646, 1:500), rabbit polyclonal anti-COPB1 (CUSAB CSB-PA005783LA01HU, 1:500), mouse monoclonal anti-Lamin A/C (Santa Cruz sc-7292, 1:500), rabbit polyclonal anti-NCDN (Proteintech 13187-1-AP, 1:500), mouse Monoclonal anti-tubulin (Sigma Aldrich, 1:500) and rabbit polyclonal anti-Histone H3 (Proteintech 17168-1-AP, 1:300).

Techniques: Western Blot, Affinity Purification, Immunoprecipitation, Mass Spectrometry, Sequencing

Journal: bioRxiv

Article Title: Neurochondrin interacts with the SMN protein suggesting a novel mechanism for Spinal Muscular Atrophy pathology

doi: 10.1101/183640

Figure Lengend Snippet: A) Affinity isolation of NCDN-GFP using GFP-Trap, detected with anti-GFP (top row) co-enriches mCherry-SmB, detected with anti-mCherry (bottom row) in transiently co-transfected SH-SY5Y cells B) In an SH-SY5Y cell line constitutively expressing NCDN-GFP, affinity isolation of NCDN-GFP, detected with anti-GFP (top row) co-enriches SMN, SmB, SmN and the Coatomer protein βCOP, all detected with antibodies against the endogenous proteins (as labelled). C) In an SH-SY5Y cell line constitutively expressing YFP, affinity isolation of YFP, detected with anti-GFP (top row) does not co-enrich SMN, SmB or βCOP, all detected with antibodies to the endogenous proteins (as labelled). D) in an SH-SY5Y cell line constitutively expressing GFP-SMN, affinity isolation of GFP-SMN, detected with anti-GFP (top row) co-enriches NCDN, detected with anti-NCDN (middle row). Endogenous SMN, detected with anti-SMN (bottom row) is also co-enriched. E) NCDN-GFP and mCherry-SmN co-localise in vesicle-like structures (arrows) in neurites of SH-SY5Y cells constitutively expressing mCherry-SmN, and transiently expressing NCDN-GFP (left hand panels). White areas in the merged image show areas of co-localisation. Co-localisation images (bottom row) were generated in Volocity, using automatic thresholds on undeconvolved z-sections before excluding values below 0.05 (see material and methods). No co-localisation is seen in the same cell line transiently expressing YFP alone (right hand panel). Triangles show structures containing mCherry-SmN, but not YFP. F) mCherry-SMN and NCDN-GFP co-localise in vesicles (arrows) in the cytoplasm of co-transfected SH-SY5Y cells (left hand panels). White areas in the merged image show areas of co-localisation. Co-localisation images (bottom row) were generated as above. No co-localisation is observed between mCherry-SMN and YFP (triangles, right hand panels). Bar= 7μm.

Article Snippet: Antibodies used were rat monoclonal anti-RFP (Chromotek 5F8, 1∶500); goat polyclonal anti-γCOP (Santa Cruz sc-14167, 1∶250), rabbit polyclonal anti-GFP (Abcam ab290, 1:2000), rabbit polyclonal anti-SNRPN (SmN) (Proteintech 11070-1-AP, 1:800), mouse monoclonal Y12 anti-Smith (SmB) (Abcam ab3138, 1:100), rabbit polyclonal anti-SMN (Santa Cruz sc-15320, 1:500), mouse monoclonal anti-SMN (BD Transduction labs 610646, 1:500), rabbit polyclonal anti-COPB1 (CUSAB CSB-PA005783LA01HU, 1:500), mouse monoclonal anti-Lamin A/C (Santa Cruz sc-7292, 1:500), rabbit polyclonal anti-NCDN (Proteintech 13187-1-AP, 1:500), mouse Monoclonal anti-tubulin (Sigma Aldrich, 1:500) and rabbit polyclonal anti-Histone H3 (Proteintech 17168-1-AP, 1:300).

Techniques: Isolation, Transfection, Expressing, Generated

Journal: bioRxiv

Article Title: Neurochondrin interacts with the SMN protein suggesting a novel mechanism for Spinal Muscular Atrophy pathology

doi: 10.1101/183640

Figure Lengend Snippet: Endogenous SmB and SmN fractionate similarly to YFP-SmB and YFP-SmN, with both present in the 100,000 RCF vesicle fraction in addition to being highly enriched in the nuclear pellet of fractionated SH-SY5Y cells. The gap between bands in the whole cell lysate and cell fractions signifies omitted lanes.

Article Snippet: Antibodies used were rat monoclonal anti-RFP (Chromotek 5F8, 1∶500); goat polyclonal anti-γCOP (Santa Cruz sc-14167, 1∶250), rabbit polyclonal anti-GFP (Abcam ab290, 1:2000), rabbit polyclonal anti-SNRPN (SmN) (Proteintech 11070-1-AP, 1:800), mouse monoclonal Y12 anti-Smith (SmB) (Abcam ab3138, 1:100), rabbit polyclonal anti-SMN (Santa Cruz sc-15320, 1:500), mouse monoclonal anti-SMN (BD Transduction labs 610646, 1:500), rabbit polyclonal anti-COPB1 (CUSAB CSB-PA005783LA01HU, 1:500), mouse monoclonal anti-Lamin A/C (Santa Cruz sc-7292, 1:500), rabbit polyclonal anti-NCDN (Proteintech 13187-1-AP, 1:500), mouse Monoclonal anti-tubulin (Sigma Aldrich, 1:500) and rabbit polyclonal anti-Histone H3 (Proteintech 17168-1-AP, 1:300).

Techniques:

Journal: bioRxiv

Article Title: Neurochondrin interacts with the SMN protein suggesting a novel mechanism for Spinal Muscular Atrophy pathology

doi: 10.1101/183640

Figure Lengend Snippet: A) Transfection of SH-SY5Y cells constitutively expressing GFP-SMN (Green) with siRNAs shows an increase in the number of nuclear gems (arrows) in cells transfected with siRNAs against NCDN or SmB and a decrease in the number of nuclear gems in cells transfected with siRNAs against SMN in comparison to cells transfected with non-targeting siRNAs (siControl) or siRNAs against lamin A/C as a' targeting' control. Transfection of SH-SY5Y cells with a plasmid to express GFP-SMN∆7 also results in increased numbers of nuclear gems. Cell nuclei are stained with Hoescht 33342 (grey on images). Transfection efficiency with siRNAs was greater than 90%, measured by transfection with siGlo Cyclophillin B (not shown). Bar=7μm. Images are deconvolved z-stacks taken with 0.2μm spacing B) Quantitation of numbers of gems per nucleus shows a significant increase following reduction of NCDN (10.2 (± 4.1) with siNCDN 18, 10.4 (± 4.9)) (Mean ± Standard Deviation) with siNCDN 19, 9.9 (± 4.1) with siNCDN 20, 9.5 (± 3.6) with siNCDN 21 and 10.7 (± 4.6) with siNCDN pooled compared to 4.4 (± 2.5) in cells treated with non-targeting siRNA (siControl) and 4.2 (± 2.3) in cells treated with siRNAs targeting lamon A/C (siLaminA/C). Reduction of SmB also shows an increase in numbers of nuclear gems (to 16.7 (± 6.8) with siSmB), while reduction of SMN leads to a decrease in numbers of nuclear gems (to 0.7 (± 1.4) with siSMN). Expression of GFP-SMN∆7 results in an increase of numbers of nuclear gems to 18.2 (± 5.3). The difference between each siNCDN and controls is statistically significant (AVOVA; P<0.0001, n=150 from 3 replicates). A Tukey post-test identified outliers (individual points marked on graph). C) Immunoblot analysis using antibodies to endogenous NCDN, SMN and SmB shows a reduction in expression of each of 40-60% compared to siControl cells, after signals were normalised to tubulin.

Article Snippet: Antibodies used were rat monoclonal anti-RFP (Chromotek 5F8, 1∶500); goat polyclonal anti-γCOP (Santa Cruz sc-14167, 1∶250), rabbit polyclonal anti-GFP (Abcam ab290, 1:2000), rabbit polyclonal anti-SNRPN (SmN) (Proteintech 11070-1-AP, 1:800), mouse monoclonal Y12 anti-Smith (SmB) (Abcam ab3138, 1:100), rabbit polyclonal anti-SMN (Santa Cruz sc-15320, 1:500), mouse monoclonal anti-SMN (BD Transduction labs 610646, 1:500), rabbit polyclonal anti-COPB1 (CUSAB CSB-PA005783LA01HU, 1:500), mouse monoclonal anti-Lamin A/C (Santa Cruz sc-7292, 1:500), rabbit polyclonal anti-NCDN (Proteintech 13187-1-AP, 1:500), mouse Monoclonal anti-tubulin (Sigma Aldrich, 1:500) and rabbit polyclonal anti-Histone H3 (Proteintech 17168-1-AP, 1:300).

Techniques: Transfection, Expressing, Plasmid Preparation, Staining, Quantitation Assay, Standard Deviation, Western Blot