Primary antibodies used in ChIP, EMSA, Western blotting and immunofluorescence.

a<p>Rabbit and goat antibodies are polyclonal. Mouse antibodies are monoclonal. All antibodies were affinity purified.</p>b<p>Antibodies were used for Chromatin immunoprecipitation (ChIP), Western blotting (WB, [200–500 ng/ml]), Immunofluorescence (IF, [10–20 µg/ml]), electrophoretic mobility shift assays (EMSA, [20–100 µg/ml]) or immunohistochemistry (IHC) (10 µg/ml).</p

Expression of Sp1 and Sp3 in human brain endothelium in vitro and in situ.

<p>Expression of Sp1 and Sp3 by immunofluorescence in hCMEC/D3 cells and primary human brain endothelium and by immunohistochemistry in blood vessels in the putamen of human brain.</p

Sequences of probes in MDR1 promoter region.

*<p><i>Homo sapiens</i>, chromosome-7 genomic contig. Reference sequence.</p

Trancription factor binding to the MDR1 promoter in brain endothelium.

<p>(A) ChIP was performed on hCMEC/D3 cells with antibodies to TFIID, Sp3, Sp1 and a control IgG. The proximal MDR1 promoter was detected by PCR, with primers spanning the region −223 to −1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048189#pone-0048189-g001" target="_blank">figure 1</a>). Full length promoter was used as a positive control (Pos.) No chromatin was used as an additional negative control (Neg.). (B) Western blotting of nuclear proteins from hCMEC/D3,(D3) or Caco-2 cells, with antibody to Sp3. Bands ‘a’ and ‘b’ correspond to the activating/suppressive variants of Sp3. Bands ‘d’ and ‘e’ correspond to non-activating variants. (C) Western blotting of nuclear proteins from hCMEC/D3 (D3), or Caco-2 cells, with antibody to Sp1. Previously reported variants of Sp1 correspond to bands ‘f’ and ‘g’. The blot was stripped and reprobed with antibody to TFIID as a positive control for loading and transfer.</p

Differential binding of transcription factors to the MDR1 promoter in brain endothelium and Caco-2.

<p>EMSA with probes E, F, J and K and nuclear proteins from hCMEC/D3 (D3) compared with Caco2. Arrows indicate bands that are strongly expressed in the Caco-2 cells, but absent or at low levels in hCMEC/D3 cells.</p

Interaction of Sp transcription factors with the MDR1 promoter.

<p>(A) EMSA with probe-C in lanes 1–9, and hCMEC/D3 nuclear protein in lanes 2–9. Protein-binding was blocked with a consensus Sp oligonucleotide, a mutated Sp oligonucleotide (Sp-mut.) and a consensus AP2 oligonuceotide (lanes 3–5). Supershifts were attempted with antibodies to Sp3, Sp1, TFIID and NFY (lanes 6–9). The nuclear protein produced five shifted bands, of which three (b,c,d) were reduced by anti-Sp3. (B) EMSA with probe-C in lanes 1–7 and Caco-2 nuclear protein in lanes 2–7. The nuclear proteins produced 4 major shifted bands (f–i). Protein-binding was blocked with consensus oligonucleotides for Sp or AP2 (lanes 3 & 4). Supershifts with specific antibodies (lanes 5–7) indicated that band ‘f’ was produced by Sp1, bands ‘g’ and ‘h’ by Sp3. Band ‘i’ was substantially reduced by all treatments.</p

Consensus dsDNA blocking oligonucleotides.

<p>Consensus dsDNA blocking oligonucleotides.</p

Blood vessel architecture and finite element model.

<p>(A) Scanning electron micrograph of a vascular corrosion cast illustrating the density and complexity of cortical vasculature in the rat. Cortical surface is to the top of the figure. Scale bar: 500 μm. From Merchan-Perez and DeFelipe, unpublished material. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172368#pone.0172368.s001" target="_blank">S1 Movie</a>. (B) The finite element model represents a cylinder of gray matter traversed from top to bottom by a small blood vessel. The blood vessel is considered to be a hollow cylinder with a diameter of 10 μm. The whole model diameter is 60 μm, and its height is 50 μm. Gray matter is assumed to be a linearly-elastic isotropic material. The body has been meshed into approximately 15,000 constant strain tetrahedral elements.</p

Three-dimensional model of a brain blood vessel and the tissue and synapses that surround it.

<p>The model in (A) and (B) represents a cylinder of brain tissue crossed by a blood vessel from top to bottom. Excitatory (glutamatergic) and inhibitory (GABAergic) synapses have been represented as green and red points, respectively. The cylinder height is 50 μm and its diameter is 60 μm. The diameter of the blood vessel at rest is 10 μm. In (A), the maximum contraction of the central part of the blood vessel has been represented (the diameter decreases to 8 μm). In (B), maximum dilation of the central part of the blood vessel has been represented (the diameter increases to 12 μm). No displacement was allowed at the external boundary of the model. The displacements of the synapses that were randomly distributed within the model are represented in (C) and (D) during maximal contraction, and dilation, respectively. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172368#pone.0172368.s002" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172368#pone.0172368.s003" target="_blank">S3</a> Movies.</p

Maximum displacement of points during blood vessel contraction or dilation.

<p>To compute these curves, 26 points were used. The first point was located on the maximum deformation point of the vessel wall (at the model mid-plane) and the rest were radially distributed at regular intervals (1 μm) until the external boundary of the model was reached. The maximum displacement of the blood vessel wall was 1 μm from the resting state during contraction (inwards) or dilation (outwards). No displacement in the external boundaries of the model was allowed. (A) Maximum absolute displacement of points at regular time intervals (T = 1 to T = 5) until maximum contraction or dilation of the blood vessel is reached (T = 5). (B) Inter-point distance increment as a percentage of the resting-state distance (1 μm). Points were arranged in the same way as in (A), and the distances between each point and its outward neighbor were recorded at the same time intervals. Since the behavior of the model is symmetric, these increments represent an increase of inter-point distances during blood vessel contraction and a decrease during dilation.</p