Western blot analysis
Plants were grown on agar plates for ten days. Protein-extracts from
whole seedlings were prepared as described by Novák et al.(2018). Proteins were separated on 10% SDS-PAGE and blotted onto
nitrocellulose membranes by wet transfer. Membranes were blocked in 5%
low fat milk in TBS-T for 1 h, and probed with 1:2,000 anti-PLDα1/2
(AS12 2364, Agrisera, Sweden) in 3% low fat milk in TBS-T for 1 h as
well as 1:5,000 goat anti-rabbit (Bethyl) in 5% low fat milk in TBS-T
for 1 h. Precision plus protein WesternC standard (Bio-Rad) was used to
estimate molecular weights, and this lane was separated from the
membrane after blotting and incubated separately in Blocking reagent
(Qiagen) in TBS-T for 1 h, followed by 1:10,000 Precision Protein™
StrepTactin-HRP Conjugate (Biorad) for 1 h. For loading control, the
membrane was stained with Novex reversible membrane protein stain
(Invitrogen) according to manufacturer´s instructions.