Mabn52

The prenylation reactions were set up using 20 μg of total cell lysate, recombinant rat GGT-II (2 μM; Jena Biosciences, Jena, Germany), recombinant human RAB6A (4 μM; Jena Biosciences, Jena, Germany), and biotin-labeled geranyl pyrophosphate (B-GPP, 5 μM; Jena Biosciences, Jena, Germany) as lipid donor in prenylation buffer. All reactions were supplemented with fresh guanosine 5′-diphosphate (GDP; 20 mM; Merck Millipore, Watford, UK) and DTT (1 mM; Thermo Fisher Scientific, Loughborough, UK). Positive controls were prepared using untransduced cell lysate spiked with a recombinant REP1 protein (fish His-REP1; Jena Biosciences, Jena, Germany). The reactions were incubated for 2 h at 37°C and then stopped by addition of Laemmli sample buffer. Reaction products were subjected to SDS-PAGE and immunoblot analysis as per the protocol previously described in Patrício et al.¹⁵ (link) Membranes were incubated separately for detection of human REP1 (MABN52; 1:2,500; Merck Millipore, Watford, UK) and β-actin (AM4302; 1:50,000; Thermo Fisher Scientific, Loughborough, UK), which were detected using a horseradish peroxidase (HRP)-labeled secondary antibody (1:10,000). The incorporation of biotinylated lipid donor into the RAB6A substrate was detected by direct incubation with streptavidin-HRP (1:10,000; Thermo Fisher Scientific, Loughborough, UK).