Anti actin

The following primary antibodies were used: anti-Notch1 intracellular domain (NICD) (Cell Signaling Technology, MA, USA), anti-Notch1 extracellular domain (NEXT) (Thermo Fisher Scientific, MA, USA), anti-Hey-1 (GeneTex, CA, USA), anti-Actin (TransGen, Beijing, China). Synthetic amyloid-beta peptides 1–40 (Aβ_1–40) were purchased from Invitrogen (Invitrogen, CA, USA) and dissolved in hexafluoreisopropanol (HFIP) (Sigma, MO, USA) for 2 h at room temperature, and lyophilized peptide was dissolved in dimethylsulfoxide (DMSO).

The harvested cells were lysed with lysis buffer containing protease inhibitor cocktail (Roche, Amherst, CA, USA). Protein samples were separated by SDS–PAGE and transferred to nitrocellulose membranes. The blocking buffer was prepared with 5% nonfat dry milk in TBST buffer, and the membranes were blocked at room temperature for 1 hour and then incubated with primary antibodies in a shaker at 4°C overnight. Antibodies were anti-GSTO2 (Proteintech, 14562-1-AP), anti-HUS1 (Proteintech, 11223-1-AP), anti-INTS1 (Sigma–Aldrich, HPA021658), anti-TMEM184A (Proteintech, 25989-1-AP), anti-TMEM190 (Invitrogen, PA5-70986), and anti-Actin (TransGen Biotech, HC201-01). The membranes were incubated with the corresponding secondary antibodies (1 : 2000) for 1 hour at room temperature the next day, and finally, the proteins were visualized with chemiluminescence solution.

To detect the secretory activity of VdSCP23 in vitro, a fused fragment including the TrpC-promoter region, the coding sequence of VdSCP23-GFP and the Nos-terminator was introduced into the vector pCOM [68 (link)]. The primer pairs for its amplification are listed in Table S3. The conidia of complemented strain ECVdSCP23-GFP were collected from a shake culture maintained in complete medium (CM: 0.6% yeast extract, 0.6% casein acids hydrolysate, 1% sucrose) for 3 days. The conidia were transferred to YEPD liquid medium (1% yeast extract, 2% peptone, 2% glucose) containing cotton root and shaken at 25 °C for 24 h. Acetone at a final concentration of 20% (w/v) was used to extract total protein from the culture supernatant after storage at −80 °C overnight. The solution was centrifuged at 13,000× g for 10 min at 4 °C. The total protein was lysed by adding plant RIPA lysis buffer containing phenylmethanesulfonyl fluoride to the dried precipitate. The target proteins were detected with Western blotting using anti-GFP antibodies (TransGen Biotech, Beijing, China), and anti-actin (TransGen Biotech, Beijing, China), anti-mouse (TransGen Biotech, Beijing, China) and anti-rabbit (TransGen Biotech, Beijing, China) antibodies were used as secondary detection antibodies.