Pe conjugated anti mouse igg 115 115 164

To generate A549, H1975 and H292 KO cells for BASIGIN, two ZFN plasmids (designed by Sigma-Aldrich CKOZFN1227-1KT, CompoZr Custom ZFN) targeting BASIGIN exon 2 were transfected using JetPRIME® Transfection Reagent (Polyplus-transfection SA), according to the manufacturer's instructions. Transfected cells were detected by fluorescence-activated cell sorting (FACS), with a CD147 (MAB972, R&D Systems) primary antibody and with a PE-conjugated anti-mouse IgG (115-115-164, Jackson ImmunoResearch) secondary antibody. Negative cells for BSG were selected, sorted and cloned by dilution in 96 wells. The absence of BSG expression was confirmed by immunoblotting and only clones lacking the two alleles of BSG were analysed.

LS174T, U87 and A549 BASIGIN expressing cell lines were transfected with the ZFN designed by Sigma-Aldrich (CKOZFN1227-1KT, CompoZr Custom ZFN) targeting exon 2 of BSG as previously described [16 (link), 38 (link)]. Another LS174T BSG knockout cell line was generated with a ZFN targeting exon 7 of BSG, common to all spliced isoforms of the gene (Sigma-Aldrich, CSTZFN-1KT, CompoZr Custom ZFN). Transfected cells were detected with a CD147 (MAB972, R&D Systems) primary antibody and with a PE-conjugated anti-mouse IgG (115-115-164, Jackson ImmunoResearch) secondary antibody as described [14 (link)]. Several negative clones were detected by immunoblotting and only clones disrupted for the two alleles (DNA sequencing) were studied (cl18: BSG^−/− LS174T cells, cl8 and cl39: BSG^−/− U87 cells and cl153: BSG^−/−A549 cells).