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Total proteins were isolated from 2-day-old vegetative hyphae grown in 5 x YEG culture (5g yeast extract, 10g glucose, 1L ddH₂O) using TCA-acetone precipitation methods, and total protein concentrations were determined using the Enhanced BCA Protein Assay Kit (Beyotime, China). Protein extracts (50μg) were subjected to 12% SDS-PAGE (EpiZyme, China) and transferred to a PVDF membrane as described in previous research (Zhang et al., 2018 (link)). Primary antibodies, including anti-Phospho-p44/42 MAPK antibody #4370 (Cell Signaling Technology, Inc.), anti-ERK1/2 MAPK antibody (C-9): sc-514,302 (Santa Cruz Biotechnology, Inc.), and anti-GAPDH R1208-3 antibody (HUABIO, China), along with peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies (Beyotime, China), were used in this study. An ECL chemiluminescent kit (Bio-Rad, United States) was used for Western blot detection.

The samples of HUVECs were rinsed with phosphate-buffered saline (PBS) and then add 50 μl lysis buffer (Beijing Solarbio Science & Technology Co., Beijing, China) containing RIPA buffer, 1% PMSF and 0.1% protease inhibitor cocktail in 24-well plate at 4 °C for 20 min, and collected in the 1.5 ml tube. And subsequently, the tube was centrifuged for 15 min at 12000 rpm at 4 °C. The concentration of protein used BCA assay kit (Beijing Solarbio Science & Technology Co., Beijing, China). The sample proteins were separated by 12% SDS-PAGE (EpiZyme, Shanghai, China), and then transferred on the polyvinylidene fluoride (PVDF) membrane (Merck Millopore, USA). The membrane was blocked with 5% defatted milk at room temperature for one hour. After this, the membrane was incubated by Rabbit Anti-phospho-AKT1 (Thr34) polyclonal antibody (Bioss, Beijing, China) with a dilution of 1 : 1000 and incubated by GAPDH (Cell Signaling Technology, USA) with a dilution of 1 : 1000. In the next step, the membrane was rinsed three times using Tris–HCl buffered saline with Tween 20 (TBST), and it was incubated by each corresponding secondary antibody and washed three times. Subsequently, the membrane was visualized using a Fusion FX7 Multifunction imaging system (Vilber, France). And the p-AKT and the GAPDH band were analyzed using Image J (Java 1.8.0-172).

Proteins were extracted, and the concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo). Proteins were denatured at 100 °C for 10 min. After the proteins were separated by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE, Epizyme, Shanghai, China), the gels were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore) and run at 250 mA for 90 min. Subsequently, the membranes were cut, blocked (5% skim milk powder), and incubated with diluted primary antibodies at 4 °C overnight. The primary antibodies were as follows: anti-GAPDH (ZEN-BIO 200306-7E4), anti-β-actin (ZEN-BIO 250132), anti-vinculin (ZEN-BIO R26085), anti-NR1D1 (ab174309), anti-NR1D2 (ab251948 and Protein Tech 13906-1-AP), anti-FOXM1 (CST20459), anti-LXRα (ab41902), anti-CCNB1 (CST12231), anti-CCNB2 (ab185622), anti-CENPA (CST2186), anti-CENPF (CST58982), anti-CDK1 (ZEN-BIO 200544), anti-survivin (CST2808), and anti-ARNTL (Protein Tech 14268-1-AP). The membranes were washed and incubated with secondary antibodies for 1 h according to the primary antibody sources. Immunoreactivity was visualized using enhanced chemiluminescent (ECL) chromogenic substrate (Millipore). The membranes were finally detected by using a ChemiDoc MP Imager System (Bio-Rad).