Western blot
Total plant leaf protein was extracted using RIPA lysis buffer
(ThermoFisher, MI, USA) and the protein concentration of the extracted
samples was measured using the Pierce™ BCA Protein Assay Kit. Cell
lysates were loaded on SDS-polyacrylamide gels and electrophoretically
separated. Proteins were transferred to PVDF membranes, blocked with 3%
BSA for 0.5 h at room temperature, and incubated with primary antibodies
overnight at 4 °C. After washing 3 times with TBST, the cells were
incubated with the secondary antibody for 1 h at room temperature.
Signal was detected with the aid of a chemiluminescence instrument using
standard ECL kits.