Electrophoretic transfer cell

Samples (40 μl of protein and 10 μl loading buffer) were boiled for 10 min. Protein samples were separated by 12% SDS-PAGE and transferred onto a nitrocellulose membrane for 35 min in an electrophoretic transfer cell (Bio-Rad Laboratories). Membranes were washed three times for 5 min in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% [v/v] Tween 20, pH 7.4), blocked with 5% skim milk for 2 h, and then incubated overnight at room temperature with rabbit serum (diluted 1:100 with 0.01 M PBS). Next, the membranes were washed four times for 5 min each in TBST, then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (diluted 1:1000) for 2 h. The membranes were washed four times with PBS, and protein signals detected using diaminobenzidine reagent (TIANGEN, Beijing, China).

A Mammalian Protein Extraction Kit (Solarbio, Beijing, China) was used to extract crude proteins from adult worms. Proteins were separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane using an electrophoretic transfer cell (Bio-Rad, Hercules) for 30 min. After washing with TRIS-buffered saline containing Tween-20 (TBST), membranes were incubated with 5% (w/v) skimmed milk at 37 °C for 2 h and incubated at 4 °C with polyclonal antibodies (1:200 v/v dilution) or serum from infected goats (1:200 v/v dilution) for 12 h, then with a 1:1,000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or rabbit anti-goat IgG (Boster, Wuhan, China) for 2 h. Signals were measured using an Enhanced HRP-DAB Chromogenic Substrate Kit (Tiangen).

Sodium dodecyl sulfate (SDS) sample buffer (0.5 M Tris-HCl, pH 6.8, 20% sucrose, 10% SDS, 1% bromophenol blue) was added to the concentrated samples. After equal amounts of protein (5 μg/lane) were separated on 4–20% gradient polyacrylamide gels (Bio-Rad, Hercules, CA, USA) under nonreducing conditions, the gels were blotted onto nitrocellulose membranes (Pierce, Waltham, MA, USA) in an electrophoretic transfer cell (Bio-Rad). Blots were blocked with 5% non-fat milk at 4 °C overnight and then probed with Anti-Collagen I antibody (1:1000; Abcam, Cambridge, UK, ab23446), anti-CD63 antibody (1:1000; Abcam, ab59479), anti-Hsp70 antibody (1:1000; Abcam, ab2787) and anti-CD81 antibody (1:1000; Abcam, ab59477) at room temperature for 1 h. After three washes with PBS-T (pH 7.4 and 0.1% Triton), the membrane was incubated with sheep anti-mouse IgG conjugated to HRP (NeoBioscience, Shenzhen, China) for 1 h at room temperature, followed by three washes in PBS-T. The band density was determined by Bio-Rad Quality One Software.

The gel as the result from electrophoresis process of S. scabiei protein using SDS-PAGE, continued by Western Blot. The procedure of Western Blot was performed according to existing protocols (13 ). The antigenic protein of S. scabiei was detected by SDS-PAGE and transferred to a nitrocellulose membrane for 1 h in an electrophoretic transfer cell (Bio-Rad, USA).

For identification of vesicular markers, protein extracts were isolated from EVs using RIPA protein lysis buffer containing 1 mM PMSF. Equal amounts of protein (10 μg/lane) were subjected to SDS-PAGE under non-reducing conditions, and the gels were blotted onto nitrocellulose membranes (Pierce, Waltham, MA, United States) in an electrophoretic transfer cell (Bio-Rad). The membrane was blocked with 5% non-fat milk at 4°C overnight and then probed with anti-CD63 (1:800 dilution; Abcam), anti-CD9 (1:800 dilution; Abcam), and anti-ALIX (1:800 dilution; Abcam) antibody for 1 h. The membrane was incubated with sheep anti-mouse IgG conjugated to HRP (NeoBioscience, Shenzhen, China) for 1 h. Band density was determined using the Bio-Rad Quality One Software. To detect the expression of key proteins in ORSCs and MxCs, total protein was extracted from human HF using RIPA lysis buffer and subjected to western blot analysis as described above. Antibodies against the following proteins were obtained from Abcam (Cambridge, United Kingdom): β-catenin (1:100 dilution), Lef-1 (1:100 dilution), Shh (1:100 dilution), Gli1 (1:100 dilution), Cyclin D1 (1:400 dilution), Cyclin E (1:400 dilution), BMP2 (1:800 dilution), and SMAD5 (1:400 dilution). p-SMAD5 was obtained from Millipore (1:800 dilution).

The protein was extracted from the developing Zea mays seeds according to the protocol described in [45 (link)]. SDS-PAGE was performed using a Vertical Electrophoresis System (Bio-Rad, Shanghai, China). Proteins were separated in SDS-PAGE on 10% acrylamide gels. Anti-rabbit antibodies (ZmPHO1) were generated according to the protocol described in [28 (link)]. After electrophoresis, the proteins in polyacrylamide gels were transferred to nitrocellulose membranes using an Electrophoretic Transfer Cell (Bio-Rad). The transfer buffer contained 10% running buffer, 20% methanol, and 70% water. After this membrane was incubated in blocking buffer (5% solution of skim milk in 1% TBST) for one hour on a rotator and then incubated overnight at 4 °C. A 15 µL quantity of ZmPHO1 antibody (affinity pure antibody) was added with (Anti-PHO1) 1:1000 dilution in blocking buffer, then incubated for 1 h on rotator at room temperature. The gel membrane was washed three times for 10 min each in 1% TBST. HPR secondary binding antibody Rabbit IgG was added with 1:2000 dilution in blocking buffer. The gel membrane was incubated for one hour. The gel membrane was washed three times for 10 min each in 1% TBST. A resolving solution was added according to the company or manufacturer’s instruction to photograph the bands’ results.

Protein extracts were prepared by homogenizing coenurus in an NP-40 cell lysis buffer (Boster, Wuhan, China). Purified rTmP2 proteins and total worm extract were separated by SDS-PAGE and transferred onto Polyvinylidene Fluoride membranes (Boster) for 30 min in an electrophoretic transfer cell (Bio-Rad, USA). The membrane was blocked with 5 % skim milk in Tris Buffered Saline with Tween-20 (TBST) for 2 h at room temperature. Membranes were then incubated overnight at 4 °C with goat antiserum from naturally infected goats (diluted 1:100 (v/v) in 1 % skim milk in TBST). And the rest of the program was performed as described previously [34 (link)].