The carboxyterminal part of dynactin-1-p150Glued was RT-PCR-cloned from human glioblastoma cDNA using primers attB1-dyn 2532 (5′-AAA AAG CAG GCT TCA CCA TGG CAG CTG CTG CTG CC-3′, sense) and attB2-dyn 3837 (AGA AAG CTG GGT GTT AGG AGA TGA GGC GAC TGT G-3′, antisense). AttB1 and attB2 sites were extended in a second PCR and products were cloned via pDONR201 in p3xflg-CMV (Invitrogen, Carlsbad, CA, USA) to generate p3xflg-CMV-dyn 2532–3837. Plasmids were transfected into COS1 cells in 8-well glass slides (Lab-Tek Chamber Slide™ System, Nunc, Roskilde, Denmark). Forty-eight hours later, cells were fixed with ice-cold acetone and incubated with nanobody C-C7 for 1 hr, followed by sequential incubations with mouse anti-VSV and goat-anti-mouse FITC (Invitrogen, Carlsbad, CA, USA). After washing, slides were stained with rabbit-anti-dynactin-1 which was detected with donkey-anti-rabbit TRITC (Invitrogen, Carlsbad, CA, USA). Images were processed on a Leica Discovery Fluorescence Microscope and a Leica confocal microscope.
van Lith S.A., Roodink I., Verhoeff J.J., Mäkinen P.I., Lappalainen J.P., Ylä-Herttuala S., Raats J., van Wijk E., Roepman R., Letteboer S.J., Verrijp K, & Leenders W.P. (2016). In vivo phage display screening for tumor vascular targets in glioblastoma identifies a llama nanobody against dynactin-1-p150Glued. Oncotarget, 7(44), 71594-71607.
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