Plexind1

Total cell lysates were prepared in a Tris pH 6.8–10% SDS solution (1:1) by heating at 95° for 20 mins. Protein concentration was measured using Pierce BCA protein assay kit as per the company instructions. For western blotting 10–40 ug of protein was resolved on 7.5% or 10% mini gels from Biorad, transferred to nitrocellulose membrane using semi-dry method and immunoblotted. 10% BSA was used for filter blocking in all conditions.
The following primary antibodies were used: against Notch1, Slug and Actin (Santa Cruz Biotechnology), against PlexinD1 (R&D Systems), against Vinculin (Sigma), against Notch3 (Cell Signaling technology), against E-cadherin (BD Transduction laboratories).

Samples for Nrp1, plexin A2, plexin A4, and plexin D1 endogenous expression in Cos-7 cells were collected 24 h after passaging. Samples for transient expression of Nrp1-eGFP, plexin A2-eGFP, plexin A4-eGFP, and plexin D1-eGFP in Cos-7 cells were transfected with 2.5 μg plasmid DNA 24 h after passaging and collected 24 h posttransfection. Cells were lysed using radioimmunoprecipitation assay lysis buffer supplemented with protease inhibitors (benzamidine, leupeptin, and PMSF). Western blotting experiments with these samples were carried out to confirm the expression of the samples. Primary antibodies used are Nrp1 (Cell Signaling; catalog no. 3725), plexin A2 (R&D; catalog no. MAB5486), plexin D1 (R&D; catalog no. AF4160), and plexin A4 (R&D; catalog no. MAB5856).