Protease inhibitor mixture

In short, cells were collected and lysed using RIPA protein extraction reagent (Solarbio) and protease inhibitor mixture (Solarbio). The same amount of protein was electrophoresed on an 8% SDS-PAGE gel, then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA), and then degreasing in a buffer (5% in TBST Blocked in milk) and then incubated with anti-rabbit ZEB1 antibody (1:1000 dilution, Cell Signaling Technology) and anti-rabbit ZEB2 antibody (1:2500 dilution, Proteintech) at 4 °C for 12 hours. Anti-rabbit GAPDH antibody (1:5000 dilution, Proteintech) was used as a loading control. Horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG antibody (1:5000, Proteintech) was used as the secondary antibody.

Cells were treated with the above concentrations of FB₁ for 48 h, then washed three times with ice-cold PBS. The cells were then lysed with RIPA buffer (Solarbio) containing a protease inhibitor mixture (Solarbio, Beijing, China) and protein phosphatase inhibitor (Solarbio). The total protein concentrations were determined using a Bradford protein kit (Tiandz, Beijing, China). The total proteins were resolved by electrophoresis and transferred to poly(vinylidene fluoride) membranes for 80 min at 200 mA. PVDF membranes were incubated with primary antibodies caspase3 (Beijing Biosynthesis Biotechnology Co., Ltd. Beijing, China), caspase9 (Bioss), CDK2 (Bioss), CDK4 (Bioss, Beijing, China), cyclinD1 (immunoway, Plano, TX 75024, USA), cyclinE1 (immunoway), Bax (Proteintech Group, Inc., Wuhan, China), and GAPDH (Servicebio, Wuhan, China) overnight at 4 °C. Then, the membrane was washed three times with Tris-buffered saline tween (TBST) for 15 min. Secondary antibody (IgG, Vazyme) was used to incubate the membranes for 1 h at 4 °C. Finally, band density was quantified using the Image Lab 5.0 on the ChemiDOC XRS + system (Bio-Rad, Hercules, CA, USA).

Total protein was extracted from brain tissue or cultured cells with ice-cold RIPA buffer and protease inhibitor mixture (Solarbio). Equal amounts of protein per track were loaded onto 10% SDS-PAGE gels (Epizyme, Shanghai, China) for electrophoresis and then transferred onto PVDF membranes (Millipore) at low temperature. After blocking in 5% non-fat milk, the membranes were incubated overnight with primary antibody followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody. The primary antibodies included anti-GAPDH (#10494-1-AP, Proteintech), anti-beta tubulin (#10094-1-AP, Proteintech), anti-TH (#AB152, Millipore), anti-CHIP (#2080, CST; Danvers, MA, USA), anti-Opa1 (27733-1-AP, Proteintech), anti-Drp1 (12957-1-AP, Proteintech), anti-Mfn1 (#13798-1-AP, Proteintech), anti-Mfn2 (#12186-1-AP, Proteintech), and anti-Fis1 (#10956-1-AP, Proteintech). Membranes were developed using the ECL luminescence reagent (#SQ101, Epizyme) and the protein bands visualized by enhanced chemiluminescence. Signal intensities were quantified with Image J software (NIH, Bethesda, MD, USA) and normalized to an internal control protein.

The Fol-infected tissues or plants were frozen and ground in liquid nitrogen and then homogenized in protein extraction buffer as described previously (Xian et al., 2020 (link)). The culture supernatant protein was extracted as described previously (Li et al., 2020a (link)). To determine that FolSvp1 could translocate to host roots, tomato roots were inoculated with a spore suspension (5×10⁶ spores/ml) of the FolSvp1-GFP-overexpressing transformants for 24 hr, and subsequently used for protoplast collection and protein extraction as described previously (Ji et al., 2021 (link)). Plant cytoplasm or nuclear proteins were extracted from agroinfiltrated N. benthamiana leaves using a Nuclear Extraction Kit (Solarbio) and a protease inhibitor mixture (Solarbio) 3 days after infiltration following the manufacturer’s instructions. Apoplastic fluid from agroinfiltrated leaves was isolated as described previously (Dong et al., 2014 (link); Shabab et al., 2008 (link)).

The plant tissues were frozen and ground in liquid nitrogen and then homogenized in a protein extraction buffer and a protease inhibitor mixture (Solarbio) as described previously (Xian et al., 2020 (link)). The obtained proteins were separated by 12% SDS-PAGE gel and immunoblotted using anti-GFP (1:5000 dilution, Abcam, ab183734). The signals in the blots were visualized with ECL kits (Millipore) and photographed using an Amersham Imager 680 (GE Healthcare). SDS-PAGE was performed with Coomassie brilliant blue (CBB) straining to verify equal loading.

Treated HCM cells were lysed on ice using radioimmunoprecipitation assay lysis buffer (Beyotime, China) with protease inhibitor mixture (Solarbio, Beijing, China) (100:1). The protein concentrations were determined using a bicinchoninic acid protein assay kit (Solarbio, Beijing, China). Total protein (20 μg) was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were then blocked with 5% non-fat dry milk for 2 h at room temperature and incubated at 4°C overnight with primary antibodies. Rabbit anti-MAPK14 (1:1000, Abcam, San Francisco, CA, USA) and rabbit anti-tubulin (1:1000) (CST, Danvers, MA, USA) antibodies were used for probing. After incubation, membranes were washed three times with 1× TBST for 10 min and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:1000, Proteintech, China) for 1 h at room temperature. Protein bands were detected using enhanced chemiluminescence (Amersham Imager 600).

Cells were rinsed twice in ice-cold PBS. The RIPA buffer (Solarbio), protease inhibitor mixture (Solarbio), and protein phosphatase inhibitor (Solarbio) were subsequently added to the extract protein. Supernatants were used to quantify the protein concentration via the Bradford protein kit (Tiandz, Beijing, China). Proteins were separated by sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V for 40 min and 120 V for 50 min, and subsequently electrotransferred to polyvinylidene fluoride (PVDF) membranes at 200 mA for 80 min. Membranes were incubated with primary antibody P21 (Proteintech, Wuhan, China), P53 (Bioss, Beijing, China), PCNA (Bioss), p-Cyclin B1 (Bioss), Claudin1 (Bioss), Occludin (Bioss), ZO-1 (Bioss), Cdc25c (CST, Boston, MA, USA), and GAPDH (Servicebio, Wuhan, China) overnight at 4 °C. After incubation with corresponding secondary antibodies, the protein band densities were quantified using Bio-Rad ChemiDoc™ XRS and Image Lab™ Software.