As003

The cultured cells were collected and lysed with enhanced radioimmunoprecipitation assay lysis buffer (Boster Biological Technology, Wuhan, P.R. China) containing protease inhibitors. The bicinchoninic acid kit (Boster Biological Technology) was utilized to determine the protein concentration. Proteins were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the separated proteins were electro-transferred to polyvinylidene fluoride membrane (Immobilon P, Millipore, Billerica, MA, USA), and the membrane was treated with 5% skimmed milk at ambient temperature for 2 h to block non-specific binding. The membrane was incubated with primary antibody at 4°C overnight and then with horseradish peroxidase conjugated secondary antibody at 37°C for 1 h. Enhanced chemiluminescence reagent (Thermo Fisher Scientific) was utilized to visualize the immunoreactive bands, followed by imaging using ChemiDoc XRS Plus luminescent image analyzer (Bio-Rad, Hercules, CA, USA). ImageJ analysis software was utilized to quantify the gray value of protein bands. Antibodies included SIRT6 (A7416, 1:2,000, ABclonal, Woburn, MA, USA), NRP-1 (A19087, 1:2,000, ABclonal), GAPDH (AC033, 1:50,000, ABclonal), Lin28B (ab191881, 1:2,000, abcam), rabbit secondary antibody (AS014, 1:10,000, ABclonal), and murine secondary antibody (AS003, 1:10,000, ABclonal).

The indicated strains were inoculated into liquid MM and cultivated for 24 hours at 37°C and 200 rpm. With a mortar and pestle, the mycelia were gathered and homogenized in liquid nitrogen. The protein extracts were obtained as previously described [58 (link)]. The nuclear and cytoplasmic fractions were extracted by using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0027). SDS–PAGE gels were used to separate the total protein, which was then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Anti-mouse FLAG (1:5000, Sigma–Aldrich, F3165), anti-rabbit H3 (1:5000, Sigma–Aldrich, H9289), anti-mouse GFP (1:2000, Roche, 11814460001) and anti-rabbit H3K14ac (1:5000, Abclonal, A7254) were used to probe the blots. The secondary antibodies were peroxidase-labeled goat anti-mouse (1:5000, ABclonal, AS003) and goat anti-rabbit (1:5000, ABclonal, AS014). The Enhanced ECL luminescence detection kit (Vazyme, E411) was used to visualize blots, and images were captured with a Tanon 4200 chemiluminescent imaging system. ImageJ software was used to calculate the band intensities.

Total protein was extracted by lysis of cells with SDS-PAGE Sample Loading (P0015, Beyotime), and 20 μl/ lane of protein extract was separated on SDS–polyacrylamide gels and then transferred onto PVDF membranes (0.22 µm pore, Roche). After that, the membranes were blocked with TBST buffer (20 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH8.0) containing 5% nonfat milk for 3 h, then coated with primary antibody and placed at 4 °C overnight and after that were incubated at room temperature for 1 h. Protein bands were visualized with Immobilon Western chemiluminescence HRP substrate.
Primary antibody information: β-actin(3700, CST), P62(88588, CST), LC3-A/B(12741, CST), cathepsin B(31718S, CST), cathepsin D(2284, CST), cathepsin D(AF1014, R&D), cathepsin L(AF952, R&D), PARP(5625, CST), JNK(9258, CST), P-JNK(4668, CST), P-P38(4511, CST), P38(8690, CST), Caspase 3 (GTX110543,GTX). All were diluted 1:1500.
Secondary antibody information: anti-mouse antibody (AS003, Abclonal) and anti-rabbit antibody (AS014, Abclonal), both were diluted 1:5000.