Western Blot analyses (Gel electrophoresis and immunoblotting)
Protein (5 µg per lane in plasma membrane fractions and 15 µg for
microsomal fractions) was loaded for 12% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (Yepes-Molinaet al. , 2020). The antibodies used were against Arabidopsis
thaliana PIP1 group, PIP2 (PIP2-1, PIP2-2, and PIP2-3) and PIP2-7
(Agrisera, Vännäs, Sweden). After checking predicted reactivity against
broccoli proteins, the fact that PIP1 antibody had a reactivity against
BoiPIP1-1a, BoiPIP1-2a, BoiPIP1-2b, BoiPIP1-2c, BoiPIP1-3a, BoiPIP1-4a,
BoiPIP1-4b, and BoiPIP1-5a were determined, whereas PIP2 antibody had a
predicted reactivity against (BoiPIP2-1a, BoiPIP2-1b, BoiPIP2-2a,
BoiPIP2-3a, BoiPIP2-3b, BoiPIP2-4a, BoiPIP2-4b, BoiPIP2-4c, BoiPIP2-5a,
PIP2-5b), while PIP2-7 was specific against BoiPIP2-7 isoforms. Goat
anti-rabbit IgG coupled to horseradish peroxidase was used as the
secondary antibody. A chemiluminescent signal was developed using
SuperSignal West Pico PLUS substrate (Thermoscientific., Rockford, IL,
USA). The intensity of each band was determined by ImageJ software
(Schneider et al. , 2012).