As11 1630

Seed extracts were prepared as previously described (Piskurewicz et al., 2008 (link)). Polyclonal anti-RGL2 and anti-ABI5 were as previously described (Piskurewicz et al., 2008 (link)). Polyclonal anti-GAI was produced as described in Piskurewicz et al. (2008) (link). Anti-RGA antibody was purchased (Agrisera, product AS11 1630, RRID:AB_10749442). A commercial anti-UGPase antibody was used as a loading control (Agrisera, product AS05 086, RRID:AB_1031827).

Seeds or seedlings were ground directly in 1× or 2× Laemmli loading buffer, respectively, microfuged 10 min at 4 °C to pellet debris, then boiled 5 min prior to fractionation by SDS-PAGE (10% polyacrylamide). Proteins were transferred to nitrocellulose filters, as described in [32 (link)]. Filters were blocked with Casein blocking buffer (LI-COR Biosciences, Lincoln, NE, USA), then co-incubated with anti-GFP mAb (1:10,000, UBPBio, Aurora, CO, USA) and anti-RGA pAb (1:2000, AS11 1630, Agrisera, Vännäs, Sweden) primary antibodies, followed by anti-mouse and anti-rabbit secondary IRDye 800 conjugated IgGs, and visualized using the 800 channel of the Licor Odyssey Infrared Imaging System or the iBright FL1500 Imaging System (Invitrogen, ThermoFisher Scientific, Waltham, MA USA). Filters were subsequently probed with anti-ABI5pAb (1:10,000, Ab98831, AbCam, Cambridge, UK) and anti-actin mAb (A0480, Sigma, St. Louis, MO, USA), followed by anti-mouse secondary IRDye 800 conjugated IgGs (LI-COR Biosciences, Lincoln, NE, USA).