Journal: Scientific Reports

Article Title: The transcriptome of mouse central nervous system myelin

doi: 10.1038/srep25828

Figure Lengend Snippet: ( A ) Scheme illustrating the biochemical purification of a light-weight membrane fraction enriched for myelin by homogenizing mouse brains in 0.32 M sucrose, sequential sucrose density gradient centrifugation, and osmotic shocks. Myelin accumulates at the interface between 0.32 M and 0.85 M sucrose. ( B ) Immunoblot analysis of myelin-enriched fractions and equal amounts of brain lysate to compare the abundance of marker proteins for compact myelin (PLP/DM20, MBP), non-compact myelin (CNP, SIRT2), the oligodendroglial nuclear/cytoplasmic compartment (OLIG2), astrocytes (GFAP), microglia (AIF1/IBA1), neuronal plasma membrane (GPM6A), axonal microtubules (TUBB3/TUJ1), and mitochondria (VDAC). Blot represents three biological replicates (male c57Bl6/N mice, age P75). Note that myelin markers were enriched in purified myelin while markers of other cellular sources were reduced. ( C ) Heatmap displaying reverse CT values from qRT-PCRs for three markers each specific for myelin, migroglia, neurons and astrocytes performed on myelin biochemically purified from the brains of 4 individual mice (M1–4) compared to the respective brain lysates (BL1–4) at six month of age.

Article Snippet: Antibodies were specific for PLP/DM20 (A431 , 1:5000), MBP (Dako A0623, 1:500), CNP (Sigma C 5922, 1:1000), SIRT2 (Abcam ab67299, 1:500), OLIG2 (DF308 , 1:200, kindly provided by J. Alberta and C. Stiles, Boston, MA, USA) GFAP (Novocastra NCL-GFAP-GA5, 1:500), AIF (also termed IBA1; Abcam ab107159, 1:500), GPM6A (#24924 , 1:1000), TUBB3 (also termed TUJ1; Covance MMS-435P, 1:1000), and VDAC (Rockland 600-401-882, 1:2000).

Techniques: Purification, Membrane, Gradient Centrifugation, Western Blot, Marker, Clinical Proteomics

Journal: Brain Sciences

Article Title: Differential Expression of Multiple Disease-Related Protein Groups Induced by Valproic Acid in Human SH-SY5Y Neuroblastoma Cells

doi: 10.3390/brainsci10080545

Figure Lengend Snippet: Pathway enrichment.

Article Snippet: Cellular protein was mixed with sample buffer (62.5 mM Tris-HCl pH = 6.8, 2% SDS, 25% glycerol, 0.01% Bromophenol Blue, 5% β-mercaptoethanol) and denatured by heating at 95 °C for 10 min. Immunoblotting analysis was performed using standard protocols with the following primary antibodies: rabbit anti-ATP5D (A9929, ABclonal, Woburn, MA, USA); rabbit anti-ATP5J (tcea21587, Taiclone, Taipei, Taiwan); rabbit anti-DPH6 (23993-1-AP, Proteintech, Rosemont, IL, USA); rabbit anti-FBXO4 (tcea12933, Taiclone); rabbit anti-FSTL1 (A15789, ABclonal); mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G8795, Sigma-Aldrich); mouse anti-HDAC1 (GTX100513, GeneTex, Hsinchu City, Taiwan); rabbit anti-HDAC2 (GTX112957, GeneTex); rabbit anti-HIST1H2B (MAB15119, Abnova, Taipei, Taiwan); rabbit anti-KCNAB2 (17890-1-AP, Proteintech); rabbit anti-NPTX2 (A12031, ABclonal); rabbit anti-SCG3 (A7799, ABclonal); rabbit anti-SMARCA4 (tcea559, Taiclone); rabbit anti-UBE1L (tcea10708, Taiclone); rabbit anti-UBE2D1 (tcea7105, Taiclone); rabbit anti-UQCRB (tcea18400, Taiclone); rabbit anti-VDAC1 (tcea521, Taiclone); rabbit anti-VSNL1 (A6999, ABclonal).

Techniques: Expressing

Journal: Brain Sciences

Article Title: Differential Expression of Multiple Disease-Related Protein Groups Induced by Valproic Acid in Human SH-SY5Y Neuroblastoma Cells

doi: 10.3390/brainsci10080545

Figure Lengend Snippet: Immunoblotting analysis to validate the differential expression for 9 proteins in VPA-treated and PBS-treated biological replicated SH-SY5Y cells. ( A ) Immunoblotting showed the expression of ATP5J, FBXO4, HDAC1, HDAC2, SMARCA4, UBE1L, UBE2D1, UQCRB, and VDAC17 in VPA-treated and PBS-treated SH-SY5Y cells. ( B ) Quantification showing the fold differences in the expression of ATP5J, FBXO4, HDAC1, HDAC2, SMARCA4, UBE2D1, UQCRB, and VDAC1 between VPA-treated groups and PBS control. GAPDH was a loading control. The data are expressed as fold change to PBS ± SD (* p < 0.05, n = 3 to 6).

Article Snippet: Cellular protein was mixed with sample buffer (62.5 mM Tris-HCl pH = 6.8, 2% SDS, 25% glycerol, 0.01% Bromophenol Blue, 5% β-mercaptoethanol) and denatured by heating at 95 °C for 10 min. Immunoblotting analysis was performed using standard protocols with the following primary antibodies: rabbit anti-ATP5D (A9929, ABclonal, Woburn, MA, USA); rabbit anti-ATP5J (tcea21587, Taiclone, Taipei, Taiwan); rabbit anti-DPH6 (23993-1-AP, Proteintech, Rosemont, IL, USA); rabbit anti-FBXO4 (tcea12933, Taiclone); rabbit anti-FSTL1 (A15789, ABclonal); mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G8795, Sigma-Aldrich); mouse anti-HDAC1 (GTX100513, GeneTex, Hsinchu City, Taiwan); rabbit anti-HDAC2 (GTX112957, GeneTex); rabbit anti-HIST1H2B (MAB15119, Abnova, Taipei, Taiwan); rabbit anti-KCNAB2 (17890-1-AP, Proteintech); rabbit anti-NPTX2 (A12031, ABclonal); rabbit anti-SCG3 (A7799, ABclonal); rabbit anti-SMARCA4 (tcea559, Taiclone); rabbit anti-UBE1L (tcea10708, Taiclone); rabbit anti-UBE2D1 (tcea7105, Taiclone); rabbit anti-UQCRB (tcea18400, Taiclone); rabbit anti-VDAC1 (tcea521, Taiclone); rabbit anti-VSNL1 (A6999, ABclonal).

Techniques: Western Blot, Expressing

Journal: Brain Sciences

Article Title: Differential Expression of Multiple Disease-Related Protein Groups Induced by Valproic Acid in Human SH-SY5Y Neuroblastoma Cells

doi: 10.3390/brainsci10080545

Figure Lengend Snippet: RT-qPCR assay showing the expression of 14 genes ( ATP5F1A , ATP5PF , BAZ1B , BMS1 , FBXO2 , HDAC1 , HDAC2 , SDHA , SMARCA4 , UBA7 , UBE2D1 , UQCRB , VDAC1 , and WDR36 ) in VPA-treated and PBS-treated SH-SY5Y cells. The GAPDH gene was used as the endogenous gene for normalization. The data are expressed as fold change to PBS ± SD (* p < 0.05, n = 3 to 6).

Article Snippet: Cellular protein was mixed with sample buffer (62.5 mM Tris-HCl pH = 6.8, 2% SDS, 25% glycerol, 0.01% Bromophenol Blue, 5% β-mercaptoethanol) and denatured by heating at 95 °C for 10 min. Immunoblotting analysis was performed using standard protocols with the following primary antibodies: rabbit anti-ATP5D (A9929, ABclonal, Woburn, MA, USA); rabbit anti-ATP5J (tcea21587, Taiclone, Taipei, Taiwan); rabbit anti-DPH6 (23993-1-AP, Proteintech, Rosemont, IL, USA); rabbit anti-FBXO4 (tcea12933, Taiclone); rabbit anti-FSTL1 (A15789, ABclonal); mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G8795, Sigma-Aldrich); mouse anti-HDAC1 (GTX100513, GeneTex, Hsinchu City, Taiwan); rabbit anti-HDAC2 (GTX112957, GeneTex); rabbit anti-HIST1H2B (MAB15119, Abnova, Taipei, Taiwan); rabbit anti-KCNAB2 (17890-1-AP, Proteintech); rabbit anti-NPTX2 (A12031, ABclonal); rabbit anti-SCG3 (A7799, ABclonal); rabbit anti-SMARCA4 (tcea559, Taiclone); rabbit anti-UBE1L (tcea10708, Taiclone); rabbit anti-UBE2D1 (tcea7105, Taiclone); rabbit anti-UQCRB (tcea18400, Taiclone); rabbit anti-VDAC1 (tcea521, Taiclone); rabbit anti-VSNL1 (A6999, ABclonal).

Techniques: Quantitative RT-PCR, Expressing