Western Blot Analysis of hCMEC/D3 Cells

Detergent extracts of the HCMEC/D3 cells and hCMEC/D3-MDR1-EGFP cells were prepared as described before21 (link) and subjected to Western blot analysis using anti-PGP 1:200 (Signet Laboratories, Dedham, MA, USA), anti-acetyl-histone-H4 1:200 (pan Lys 5, 8, 12, Merck Chemicals GmbH, Schwalbach, Deutschland) and anti-actin 1:1000 (Sigma-Aldrich) as primary antibiodies. The secondary antibodies utilized anti-mouse-HRP 1:1000 and anti-rabbit-HRP 1:1000 (Dako, Hamburg, Germany). Proteins were visualized by enhanced chemiluminescence using SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific) and the ChemiDoc system (Bio-Rad, Munich, Germany) with QuantityOne software (Bio-Rad) according to the manufacturer’s instructions. The indicated protein signals were quantified densitometrically with QuantityOne software (Bio-Rad) and calculated by normalization to the reference signals of actin with GraphPad Prism software (GraphPad, San Diego, CA, USA).

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Noack A., Noack S., Buettner M., Naim H.Y, & Löscher W. (2016). Intercellular transfer of P-glycoprotein in human blood-brain barrier endothelial cells is increased by histone deacetylase inhibitors. Scientific Reports, 6, 29253.

Publication 2016

anti-acetyl-histone-H4 anti-actin SuperSignal West Femto Chemiluminescent Substrate ChemiDoc system QuantityOne software GraphPad Prism software

Actin Antibodies Cells Cells extracts Chemiluminescence Detergent Histone h4 Mouse Pgp 1 Prism Protein Rabbit Western blot analysis

Corresponding Organization :

Other organizations : University of Veterinary Medicine Hannover, Foundation, Medizinische Hochschule Hannover

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Variable analysis

independent variables

Detergent extracts of the HCMEC/D3 cells and hCMEC/D3-MDR1-EGFP cells

dependent variables

Protein expression levels of P-glycoprotein (PGP), acetyl-histone-H4, and actin

control variables

Primary antibodies: anti-PGP 1:200, anti-acetyl-histone-H4 1:200, and anti-actin 1:1000
Secondary antibodies: anti-mouse-HRP 1:1000 and anti-rabbit-HRP 1:1000
Visualization method: Enhanced chemiluminescence using SuperSignal West Femto Chemiluminescent Substrate and ChemiDoc system with QuantityOne software
Quantification method: Densitometric analysis with QuantityOne software and normalization to actin reference signals using GraphPad Prism software

controls

Positive control: Not specified
Negative control: Not specified

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