Anti gapdh

For SDS-PAGE, 20 µL of protein sample was mixed with 5 µL of 5×Loading buffer and boiled for 10 min before loading. After electrophoresis, the gel was retained using Coomassie Brilliant Blue R-250. For western blot, all the mycelia from each condition were harvested and ground to extract crude protein with equal volumes of buffer, then the 10 µL protein sample for GAPDH, and the 3 µL 10 times diluted protein solution for PyrG-flag were loaded on 12% SDS-PAGE and then transferred to PVDF membranes. Non-specific antigen binding was blocked with TBS-T (20mM Tris, 137mM NaCl and 0.1% Tween-20) with 5%(m/V) skimmed milk powder for 1 h. Then, membranes were washed 10 min with TBS (20mM Tris, 137mM NaCl and 20% methanol). Membranes were incubated with anti-flag-tag (TransGen Biotech, China) or anti-GAPDH (TransGen Biotech, China) primary antibodies for 2 h. Membranes were washed 3 10 min washes with TBS and anti-flag-tag/anti-GAPDH membranes were further incubated with anti-mouse antibody/anti-rabbit antibody for 2 h followed by 3 10 min washes in TBS. The band of proteins were visualized with boyoECL Plus kit (Solarbio, China) and imaged using the ChemiDoc MP Imaging System (Bio-Rad).

Embryos (n = 200 embryos per pool) were lysed in radioimmune precipitation assay buffer, and proteins were separated on 10–15% polyacrylamide gels at 150 V for 70–90 min. Subsequent PVDF membrane transfers were performed at 250 mA for 50 min at 4 °C. After the transfer procedure, membranes were incubated for 1 h in blocking buffer (Tris-buffered saline, pH 7.4 (TBS), with 0.1% (v/v) Tween 20 and 5% (w/v) nonfat milk at room temperature) followed by incubation overnight with one of the following primary antibodies in blocking buffer on a rotating shaker at 4 °C: anti-H3K9me3 (1:300 dilution; Abcam, Cambridge, UK) and anti-GAPDH (1:1000 dilution; TransGen Biotech, Beijing, China). The membranes were then washed and incubated with a horseradish peroxidase-conjugated anti-rabbit-IgG secondary antibody at (1:500 dilution; Thermo Scientific, Waltham, MA) for 1 h at room temperature. Immunoreactive proteins were visualized by autography with the SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific, Rockford, IL). The protein concentrations of BFF lysates were measured using the bicinchoninic acid method. The protein separation, blotting, and visualization procedures were performed as described above.

Cells were lysed in PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Inc., Gyeonggi-do, Korea) to prepare protein lysates according to the manufacturer’s instructions. Cell lysates were centrifuged at 12,000 rpm for 10 min at 4 °C. Protein concentration was measured using Bradford Easy Protein Quantitative Kit (TransGene). Equal amounts of protein were separated in 10% polyacrylamide gels (Sigma-Aldrich) and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 10% non-fat milk and incubated with anti-α-SMA (Abcam, Cambridge, UK), anti-collagen I (Abcam), anti-E-cadherin (Abcam), anti-N-cadherin (Bioss, Beijing, China), albumin (Bioss), matrix metalloproteinase 9 (MMP9) (Bioss), vimentin (Bioss), pan-cytokeratin (a wide spectrum cytokeratin antibody, Bioss), fibronectin (Bioss), β-catenin (Sigma) and anti-GAPDH (TransGene) antibodies at 4 °C overnight. After washing the membranes with Tris-buffered saline containing Tween 20 three times for 5 min each time, the membranes were incubated with secondary antibodies. Finally, immunoreactive proteins were detected using an enhanced chemiluminescence detection kit (Beyotime, Shanghai, China).