Anti rubisco large subunit rbcl

Enzymatic activities of the Rubisco and PEPC enzymes were measured as described previously (Cousins et al., 2007 (link); Pengelly et al., 2010 (link)) with some modifications. The activity of both enzymes was calculated by monitoring the decrease of NADH absorbance at 340 nm with a spectrophotometer (Unico UV-2802PC, USA).
Immunoblotting was performed according to Cousins et al. (2007) (link) with some modifications. After SDS–PAGE, the gels were subjected to immunoblotting using antisera including anti-Rubisco large subunit-RBCL (Agrisera, Sweden, product no. AS03 037), anti-Rubisco small subunit RBCS (Agrisera product no. AS07 259), or anti-PEPC (Agrisera product no. AS09 458) of Arabidopsis thaliana. The total soluble protein content was determined according to Bradford (1976) (link).

The protoplasts were washed twice in cold PBS and lysed with Laemmli sample buffer. Proteins were separated by SDS-PAGE on 10% polyacrylamide gels and then transferred to nitrocellulose. Membranes were blocked with 5% (w/v) non-fat dried milk in Tris-buffered saline containing 0.1% (v/v) Tween 20 at room temperature for 1 h. After being blocked, membranes were incubated overnight with primary antibodies, namely, anti-IDO1 (Merck Millipore, Darmstadt, Germany), anti-GFP (Thermo Fisher Scientific, Waltham, MA, USA), and anti-Rubisco large subunit (rbcL, Agrisera, Sweden), then washed and incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. Immunoreactive bands were detected by enhanced chemiluminescence (Bio-Rad, Hercules, CA, USA).