Journal: Molecular Neurodegeneration

Article Title: MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navβ2 protein coding region in rat with chronic brain hypoperfusion

doi: 10.1186/s13024-015-0032-9

Figure Lengend Snippet: Navβ2 was decreased in hippocampi and cortices of rats after CBH. a , a model for Nav α-subunit trafficking after covalently binding to Navβ2 subunit. b , western-blot analysis of Navβ2 in sham and 2VO rats, upper: representative immunoblots of Navβ2; lower: the quantitative analysis data of the immunoblots. The optical density was evaluated for each band and values for 2VO rat tissue were normalized to sham group after correction for protein loading with β-actin. ** P < 0.01 vs sham, mean ± s.e.m, n = 6. c , The relative quantification of Navβ2 mRNA (SCN2B) levels was detected by real-time PCR with normalized to β-actin, followed by further normalization to the values from sham brain tissues. P > 0.05 vs Sham, mean ± s.e.m, n = 3. d , miR-9 level detected by TaqMan real-time PCR in hippocampi and cortices from sham and 2VO rats after normalization to U6 levels. * P < 0.05 vs sham, mean ± s.e.m, n = 3

Article Snippet: The primary anti-Nav1.1, Nav1.2, Navβ2 antibodies (Cat# ASC-001,1:200, RRID:AB_2040003; Cat# ASC-002,1:200, RRID:AB_2040005; Cat# ASC-007, 1:200, RRID:AB_2040011, Alomone Labs, Jerusalem, Israel) were used and β-actin (Kangcheng, Shanghai, China) β-actin (Kangcheng, Shanghai, China) was selected as an internal control of total proteins, mouse anti-human transferrin receptor (TfR) (Cat# 13–6800, RRID:AB_2040011, Invitrogen, USA) was selected as an internal control of surface proteins.

Techniques: Binding Assay, Western Blot, Quantitative Proteomics, Real-time Polymerase Chain Reaction

Journal: Molecular Neurodegeneration

Article Title: MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navβ2 protein coding region in rat with chronic brain hypoperfusion

doi: 10.1186/s13024-015-0032-9

Figure Lengend Snippet: miR-9 regulates SCN2B evaluated by luciferase array. a , Complementarity between miR-9 seed-matched sequence and the region coding for Navβ2 predicted by a computational and bioinformatics-based approach using RNA22/RNAhybrid. Two binding sites were found at the position of 336–358 of SCN2B CDS and the position of 575–597 of SCN2B CDS. The mutation made to genes are underlined. b , Luciferase reporter gene assay for interactions between miR-9 and its binding sites in the CDS region of the Navβ2 mRNA in HEK293T cells. Cells were transfected with luciferase-target motif chimeric vector alone, miR-9, AMO-9, or scramble negative control (NC) using lipofectamine 2000. ** P < 0.01 vs NC, mean ± s.e.m. NC-inhibitors means scramble negative inhibitor control, n = 3. c , Luciferase reporter gene assay for interactions between miR-9 and mutation of binding site in the CDS region of SCN2B in HEK293T cells, ** P < 0.01 vs NC, mean ± s.e.m, n = 3

Article Snippet: The primary anti-Nav1.1, Nav1.2, Navβ2 antibodies (Cat# ASC-001,1:200, RRID:AB_2040003; Cat# ASC-002,1:200, RRID:AB_2040005; Cat# ASC-007, 1:200, RRID:AB_2040011, Alomone Labs, Jerusalem, Israel) were used and β-actin (Kangcheng, Shanghai, China) β-actin (Kangcheng, Shanghai, China) was selected as an internal control of total proteins, mouse anti-human transferrin receptor (TfR) (Cat# 13–6800, RRID:AB_2040011, Invitrogen, USA) was selected as an internal control of surface proteins.

Techniques: Luciferase, Sequencing, Binding Assay, Mutagenesis, Reporter Gene Assay, Transfection, Plasmid Preparation, Negative Control, Control

Journal: Molecular Neurodegeneration

Article Title: MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navβ2 protein coding region in rat with chronic brain hypoperfusion

doi: 10.1186/s13024-015-0032-9

Figure Lengend Snippet: MiR-9 downregulates the expression of Navβ2 proteins. a , Verification of uptake of miR-9 by NRNs after transfection, * P < 0.05 vs NC, # P < 0.05, mean ± s.e.m, n = 3 independent RNA samples for each group. b , Effects of miR-9 on protein levels of endogenous Navβ2 in primary cultured neonatal rat neurons (NRNs), using western blot analysis. Cells were transfected with miR-9, AMO-9, miR-9 + AMO-9, or NC. mean ± s.e.m from 3 batches of cells for each group. * P < 0.05, ** P < 0.01 vs NC; ## P < 0.01 vs miR-9. c , Effects of miR-9 on SCN2B in primary cultured neonatal rat neurons (NRNs) using qRT-PCR analysis. Cells were transfected with miR-9, AMO-9, miR-9 + AMO-9, or NC. mean ± s.e.m from 3 batches of cells for each group. d , Representative confocal microscope images showing primary culture hippocampus neuron stained for Tubulin (green, upper), Navβ2 (red, middle). A merged image depicting double positivity (yellow) is shown on the bottom after transfection with miR-9 mimics or/and AMO-miR-9, negative control, n = 3. e , Repression of Navβ2 by miR-9 using the miRNA-masking antisense oligodeoxynucleotides (ODNs) techniques in NRNs determined by Western blot analysis, n = 3 batches of cells for each group, mean ± s.e.m, ** P < 0.01 vs NC; ## P < 0.01 vs miR-9, ODN1 (oligodeoxynucleotides, which masks the binding sites of miR-9, located in the position 336–358 of SCN2B CDS region); ODN-2 (oligodeoxynucleotides, which masks the binding sites of miR-9, located in the position of 575–597 of SCN2B CDS region)

Article Snippet: The primary anti-Nav1.1, Nav1.2, Navβ2 antibodies (Cat# ASC-001,1:200, RRID:AB_2040003; Cat# ASC-002,1:200, RRID:AB_2040005; Cat# ASC-007, 1:200, RRID:AB_2040011, Alomone Labs, Jerusalem, Israel) were used and β-actin (Kangcheng, Shanghai, China) β-actin (Kangcheng, Shanghai, China) was selected as an internal control of total proteins, mouse anti-human transferrin receptor (TfR) (Cat# 13–6800, RRID:AB_2040011, Invitrogen, USA) was selected as an internal control of surface proteins.

Techniques: Expressing, Transfection, Cell Culture, Western Blot, Quantitative RT-PCR, Microscopy, Staining, Negative Control, Binding Assay

Journal: Molecular Neurodegeneration

Article Title: MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navβ2 protein coding region in rat with chronic brain hypoperfusion

doi: 10.1186/s13024-015-0032-9

Figure Lengend Snippet: MiR-9 produces the disturbance of trafficking, cellular distribution of Nav1.1 and Nav1.2 in rats. a , Detection of miR-9 in hippocampi and cortices tissues after stereotaxic injection 8 weeks using qRT-PCR. Rats were transfected with lenti-pre- miR-9, lenti-pre-miR-9 + lenti-pre-AMO - miR-9, or NC. Data was shown by mean ± s.e.m from 6 rats for each group. * P < 0.05 vs NC; # P < 0.05 vs lenti-pre- miR-9. b , Navβ2 protein expressions in hippocampi and cortices tissues after treatment by lenti-pre-miR-9, lenti-pre-miR-9 + lenti-pre-AMO-miR-9, or NC for 8 weeks. c - f , Nav1.1 (C-D), Nav1.2 (E-F) total and surface protein expressions in hippocampi and cortices tissues after treatment by lenti-pre-miR-9, lenti-pre-miR-9 + lenti-pre-AMO - miR-9, or NC for 8 weeks. * P < 0.05,** P < 0.01 vs NC; # P < 0.05, ## P < 0.01 vs lenti-pre- miR-9, mean ± s.e.m, n = 6

Article Snippet: The primary anti-Nav1.1, Nav1.2, Navβ2 antibodies (Cat# ASC-001,1:200, RRID:AB_2040003; Cat# ASC-002,1:200, RRID:AB_2040005; Cat# ASC-007, 1:200, RRID:AB_2040011, Alomone Labs, Jerusalem, Israel) were used and β-actin (Kangcheng, Shanghai, China) β-actin (Kangcheng, Shanghai, China) was selected as an internal control of total proteins, mouse anti-human transferrin receptor (TfR) (Cat# 13–6800, RRID:AB_2040011, Invitrogen, USA) was selected as an internal control of surface proteins.

Techniques: Injection, Quantitative RT-PCR, Transfection

Journal: Molecular Neurodegeneration

Article Title: MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navβ2 protein coding region in rat with chronic brain hypoperfusion

doi: 10.1186/s13024-015-0032-9

Figure Lengend Snippet: AMO-miR-9 prevented the disturbed trafficking of Nav1.1/Nav1.2 induced by 2VO. a , Detection of miR-9 in hippocampi and cortices tissues after stereotaxic injection 8 weeks using qRT-PCR. 2VO rats were transfected with lenti-pre-AMO - miR-9, or NC. Data was shown by mean ± s.e.m from 6 rats for each group. * P < 0.05 vs sham; # P < 0.05 vs 2VO. b , Navβ2 protein expressions in hippocampi and cortices tissues in 2VO rats with or without lenti-pre-AMO - miR-9 treatment, or NC for 8 weeks. c - f , Nav1.1 (C-D), Nav1.2 (E-F) total and surface protein expressions in 2VO rats with or without lenti-pre-AMO - miR-9 treatment, or NC for 8 weeks. * P < 0.05,** P < 0.01 vs sham; # P < 0.05, ## P < 0.01 vs 2VO, mean ± s.e.m, n = 6

Article Snippet: The primary anti-Nav1.1, Nav1.2, Navβ2 antibodies (Cat# ASC-001,1:200, RRID:AB_2040003; Cat# ASC-002,1:200, RRID:AB_2040005; Cat# ASC-007, 1:200, RRID:AB_2040011, Alomone Labs, Jerusalem, Israel) were used and β-actin (Kangcheng, Shanghai, China) β-actin (Kangcheng, Shanghai, China) was selected as an internal control of total proteins, mouse anti-human transferrin receptor (TfR) (Cat# 13–6800, RRID:AB_2040011, Invitrogen, USA) was selected as an internal control of surface proteins.

Techniques: Injection, Quantitative RT-PCR, Transfection