SDS-PAGE and Immunoblotting of Plant PEPCs

Protein samples were denatured by heating in the presence of loading buffer (100 mm Tris–HCl, pH 8, 25% [v/v] glycerol, 1% [w/v] SDS, 10% [v/v] β‐mercaptoethanol, and 0.05% [w/v] bromophenol blue). Denatured proteins (10 μg for leaf extracts and 50 μg for root extracts) were separated by SDS‐PAGE in a Mini‐Protean® III‐2D Cell (Bio‐Rad, Hercules, CA, USA) and electroblotted onto a PVDF or nitrocellulose membrane in a semidry transfer blot system (Bio‐Rad). Polyclonal antibodies against native C₄‐photosynthetic PEPC from sorghum leaves (anti‐C₄ PTPC) were prepared as described in Pacquit et al. (1995 (link)). These antibodies recognize both photosynthetic and non‐photosynthetic PEPCs (Ruiz‐Ballesta et al., 2016 (link)). Immunolabeled proteins were detected by a chemiluminescence detection system (SuperSignal West Pico Rabbit IgGs; Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions in an Amersham Imager 600 (GE‐Healthcare, Chicago, IL, USA). The signal intensities were quantified using Image Studio™ Lite software (LI‐COR, Lincoln, NE, USA).