Shuffle t7 express competent e coli

Recombinant proteins were expressed in SHuffle T7 Express Competent E. coli (NEB) and purified using HisTrap columns from GE Healthcare followed by size exclusion chromatography, as previosly described (10 (link)). Purified monomeric proteins were validated by SDS-PAGE. Purified chondroitin sulfate A (CSA) was obtained from Sigma. Anti-V5-FITC antibodies were obtained from Invitrogen. Most cell lines were obtained from ATCC and grown in their suggested growth media with 1x penicillin and streptomycin cocktail. The Myla2059 Lymphoma cell lines were donated by Niels Ødum at the University of Copenhagen. Mice for animal studies were acquired from Taconic Biosciences.

The recombinant proteins were expressed in SHuffle T7 Express Competent E. coli (NEB) and purified using HisTrap from GE Healthcare, followed by size exclusion chromatography. Purified CSA, HS, and chondroitinase ABC were obtained from Sigma. CSC was obtained from Seikagaku, and Monoclonal anti-V5 and anti-V5-FITC antibodies were obtained from Invitrogen. Cells were transfected with siRNAs (QIAGEN) (10 nM final) against B3GAT1, CSGALNACT1, CHST11, CHST3, or ARSN using RNAiMAX (Invitrogen) and analyzed for rVAR2 binding by flow cytometry and for mRNA expression by RT-PCR.