Actin 66009 1 ig

GAS (SMB00313), CoCl₂ (232696), and calcium ionophore (A23187) were purchased from Sigma-Aldrich (St Louis, MO, USA). GAS and CoCl₂ were dissolved in phosphate-buffered solution to prepare the original solution of 100 mM, which was stored at -20° C. CQ diphosphate (HY-17589) and 3-BDO (HY-U00434) were purchased from MedChemExpress. Fluo-4AM (S1060) was obtained from Beyotime Biotechnology, Inc. (Nanjing, China). Primary antibodies against β-actin (66009-1-Ig, 1:10 000), LC3 (14600-1-AP, 1:1000), p62 (18420-1-AP, 1:1000), p62(66184-1-lg), LAMP-2 (66301-1-lg, 1:1000), and CaMKIIα (11533-1-AP, 66843-1-Ig) were purchased from Proteintech (Wuhan, China). Rabbit anti-p-p62 (Thr349) (Ab211324) was purchased from Abcam (Cambridge, MA, USA). Primary antibodies against CaMKIIα (#4436, 1:1000) and anti-p-CaMKIIα (Thr286) (#12716, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA). Goat anti-rabbit IgG (H&L)-HRP (BS13278, 1:10000) and goat anti-mouse IgG (H&L)-HRP (BS12478) were obtained from Bioworld. Goat PAb to Rb IgG Alexa Fluor®488 (Ab150077, 1:200) and goat PAb to MS IgG Alexa Fluor®647 (Ab150115) were purchased from Abcam. A fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody (A0516, 1:200) was purchased from Beyotime Biotechnology.

Soluble protein was extracted from frozen leaf material of 21-day-old plants (sixth and seventh leaf) in protein extraction buffer (50 mM HEPES-KOH pH 7.5 with 17.4% glycerol, 2% Triton X-100 and cOmplete Mini ethylenediaminetetraacetic acid (EDTA)-free Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Samples were heated at 70 °C for 15 min with 1× Bolt LDS sample buffer (ThermoFisher Scientific, UK) and 200 mM dithiothreitol (DTT). Extracts were centrifuged and the supernatants subjected to SDS-PAGE on a 12% (w/v) polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were probed with rabbit serum raised against wheat Rubisco at 1:10,000 dilution^{42 (link)}, the SSU RbcS2 from Chlamydomonas (CrRbcS2) (raised to the C-terminal region of the SSU (KSARDWQPANKRSV) by Eurogentec, Southampton, UK) at 1:1000 dilution, Actin (66009-1-Ig, Proteintech, UK) at 1:1000 dilution and EPYC1 at 1:2000 dilution^{16 (link)}, followed by IRDye 800CW goat anti-rabbit IgG (LI-COR Biotechnology, Cambridge, UK) at 1:10,000 dilution, and visualised using the Odyssey CLx imaging system (LI-COR Biotechnology), or by HRP-linked goat anti-rabbit IgG (Abcam) at 1:10,000 dilution, and visualised using Pierce ECL Western Blotting Substrate (Life Technologies).