Western-Blot Analysis and Enzyme Activity Assays

Seedling extracts for HMGR and FPS activity assays were obtained as described by Campos et al. (2014) and Arró et al. (2014), respectively. HMGR activity was determined as described by Campos et al. (2014) and is reported as picomoles of 3-HMG-CoA converted into MVA per minute per milliliter of protein extract at 37°C. FPS activity was measured as described by Arró et al. (2014) and is reported as picomoles of IPP incorporated into acid-labile products per minute per milliliter of protein extract at 37°C. Immunoblot analysis was performed as described by Keim et al. (2012) in the same protein extracts used for enzyme activity assays. Protein samples corresponding to 7 and 2 μL of each of the extracts analyzed for FPS and HMGR activity levels, respectively, were loaded into gel lanes. Rabbit polyclonal antibodies raised against FPS1 (Masferrer et al., 2002) and HMGR1 (Manzano et al., 2004) were used at 1:8,000 and 1:20,000 dilutions, respectively. The secondary antibody (goat anti-rabbit IgG conjugated to horseradish peroxidase) was used at a 1:50,000 dilution. The FPS and HMGR antibody complexes were visualized using the ECL Advance western-blotting system (GE Healthcare) according to the manufacturer’s instructions. Protein concentration was determined as described by Bradford (1976), and the amount of protein on the blotted membranes was assessed by Coomassie Blue staining.