Proteins were extracted and immunoblotting was performed as described previously (Lageix et al. 2008 (link)). After stress treatment, 1-week-old seedlings were ground in extraction buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 0.2% Triton X-100 and 1 mM DTT) containing both complete protease inhibitor and PhosSTOP phosphatase inhibitor (Roche). Extracted proteins were centrifuged, and the suspensions were incubated at 95 °C for 5 min. Proteins were separated by SDS-PAGE and then transferred to PVDF membranes for immunoblotting.
The membranes were probed using a monoclonal antibody of phospho-eIF2a (Ser51) (Catalogue no. 9721, Cell Signalling, 1/1000 dilution), a monoclonal antibody phosphor-S6K1-2 (AS132664, Agrisera, 1/5000 dilution) or an anti-HA monoclonal antibody (AS152921, Agrisera, 1/5000 dilution). After incubation with peroxidase-coupled secondary antibody (Proteintech 1/5000 dilution), the membranes were immersed in ECL Plus Western Blotting detection reagents (GE Healthcare Bio-Sciences) and finally exposed to X-film for visualization. The membranes were re-probed with anti-alpha-Actin-2 (Abmart, M20009, 1/10 000 dilution) or anti-Tubulin (AS10 681, Agrisera, 1/5000 dilution) as a loading control.
The membranes were probed using a monoclonal antibody of phospho-eIF2a (Ser51) (Catalogue no. 9721, Cell Signalling, 1/1000 dilution), a monoclonal antibody phosphor-S6K1-2 (AS132664, Agrisera, 1/5000 dilution) or an anti-HA monoclonal antibody (AS152921, Agrisera, 1/5000 dilution). After incubation with peroxidase-coupled secondary antibody (Proteintech 1/5000 dilution), the membranes were immersed in ECL Plus Western Blotting detection reagents (GE Healthcare Bio-Sciences) and finally exposed to X-film for visualization. The membranes were re-probed with anti-alpha-Actin-2 (Abmart, M20009, 1/10 000 dilution) or anti-Tubulin (AS10 681, Agrisera, 1/5000 dilution) as a loading control.
