Anti pdgf bb

Human brain microvascular ECs (ScienCell and PromoCell, isolated from adult or fetal human brains) were maintained in EC medium (ScienCell, supplemented with VEGF-A). All cells were used between passages 2 and 5. All cells were checked and showed no mycoplasma contamination. Cells were treated with recombinant human PDGF-AA (100 ng/ml, Peprotech, 100-13 A), PDGF-AB (100 ng/ml, Peprotech, 100-00AB), PDGF-BB (100 ng/ml, Peprotech, 100-14B), Ki8751 (3 nM, Bio-Techne, 228559-41-9), crenolanib (5 nM, ChemieTek, CP-868596), anti-VEGF antibody B20 (10 μg/ml, Genentech), anti-PDGF-AA (10 μg/ml, Millipore, 07-1436), anti-PDGF-BB (10 μg/ml, Millipore, 07-1437), or control rabbit IgG (10 µg/ml, Cell Signaling, 2729).

Cells were lysed with a NP-40 lysis buffer containing protease inhibitor cocktail (Roche, 11697498001), followed by measurement of total protein concentration. A total 20 μg protein of the lysates was resolved by 4–15% precast SDS-polyacrylamide gel electrophoresis gel (Bio-Rad). After transfer, polyvinylidene fluoride membranes were blotted with anti-GAPDH (Cell Signaling, 5174), anti-FSP-1 (Millipore, 07-2274, Abcam, ab27957, and Abnova H00006275-M01), anti-VEGFR-2 (Cell Signaling, 9698), anti-N-cadherin (Cell Signaling, 13116), anti-α-SMA (Abcam, ab5694), anti-NF-κB (Cell Signaling, 8242), anti-Snail (Cell Signaling, 3879s), anti-Erg (Cell Signaling, 97249), anti-Slug (Cell Signaling, 9585s), anti-PDGFR-α (Cell Signaling, 3164), anti-PDGFR-β (Cell Signaling, 3169), anti-PDGF-AA (Millipore, 07-1436), and anti-PDGF-BB (Millipore, 07-1437) antibodies at 1:1000 dilution. Proteins were detected with horseradish peroxidase-conjugated antibodies specific for either rabbit or mouse IgG (Bio-Rad), followed by ECL development (GE Healthcare, RPN2232). Uncropped scans of all immunoblots are provided in Supplementary Fig. 9.