Proteomic Analysis of Kiwifruit Extracellular Vesicles

Total proteins from TLs, EVs, and EVFs fractions of both treatment and control groups were resolved in one-dimensional SDS-PAGE and blotted onto nitrocellulose membrane. For each pollen sample (40 mg), the total protein content for EVs and EVFs fractions was loaded into the gel for both treatments. Instead, for TLs only 50 µg of total proteins were used, to avoid overloading. The membrane was incubated for 10 min with Ponceau staining to visualize protein profiles and loadings for all the fractions. The membrane was then blocked in 5% Blotting Grade Blocker (BioRad, Italy) in TBS for 30 min, and thus incubated at 4°C overnight with one of the following rabbit polyclonal antibodies: 1:2000 dilution of anti-clathrin heavy chain (Agrisera), 1:5000 dilution of anti-H+ATPase (Agrisera, Italy), 1:500 dilution of anti-COXII (Agrisera, Italy), 1:5000 dilution of anti-UGPase (Agrisera, Italy), 1:1000 dilution of anti-ARF1 (Agrisera, Italy), or 1:1000 dilution of anti-ALIX (Covalab, Italy). All membranes were then washed in TBS-Tween (0.05% v/v) and TBS, and they were incubated at room temperature for 2 h with 1:5000 goat polyclonal anti-rabbit IgG peroxidase conjugated (Sigma-Aldrich, Italy). Finally, the membranes were developed with Amersham™ ECL Prime Western Blotting Reagents (GE Healthcare, Italy) and read in chemiluminescence with Azure 280 (Azure Biosystems, California). Experiments were repeated in triplicate for each target protein. Comparison between proteins bands was performed using ImageJ (Schneider et al., 2012 (link)).
The existence of plant homologs for human ALIX in Actinidia chinensis Planch. was assessed using BLAST (Altschul et al., 1990 (link); Boratyn et al., 2012 (link)) to compare human ALIX sequence to published protein sequences in Actinidia chinensis var chinensis.