Sab4301136

Total FLS proteins were extracted using the RIPA Lysis Buffer containing protease and phosphatase inhibitors for 30 min. Then, the samples were quantified using a BCA Protein Assay kit (Beyotime Biotechnology, Shanghai, China) and then denatured at 100°C for 10 min. They were then separated using 10% sodium dodecyl sulfate- (SDS-) polyacrylamide gels and transferred into nitrocellulose membranes. The membranes were blocked using 5% BSA for 1 h. According to the different protein molecular weights, the membranes were cut into different membranes. These membranes were incubated with different primary antibodies against GAPDH (Abcam, #ab181602), SIRT3 (Abcam, #ab118334), NF-κB p65 (CST, #8242), p-NF-κB p65 (Ser536) (CST, #3033), NF-κB p65 (Acetyl K310) (Abcam, #ab19870), anti-FLAG Tag (Sigma, #SAB430113), and anti-c-Myc Tag (Sigma, #SAB4301136) at 4°C overnight. GAPDH was used as the endogenous control. All antibodies were used at a 1 : 1000 dilution. After being washed three times with TBS-T, the membranes were incubated with secondary antibodies for 1 h. After being washed another three times, the protein bands were luminesced using the Pierce™ ECL Western blotting substrate and were detected with the Bio-Rad ChemiDoc System. All assays were performed in triplicate.

The C. orbiculare strains used for Co-IP assays in vivo were CoTem1-HA, CoNpc1-MYC, CoNpc2-MYC, CoTem1-HA/CoNpc1-MYC, and CoTem1-HA/CoNpc2-MYC (see Table S2A). Total protein was extracted from vegetative mycelia using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and a protease inhibitor cocktail tablet), followed by incubation with Pierce Anti-HA magnetic beads (Thermo Fisher Scientific, USA) at 24°C for 1 h. After a washing step with RIPA buffer, the eluted proteins were separated by SDS-PAGE using a NuPAGE 4 to 12% Bis-Tris gel (Thermo Fisher Scientific) and transferred to a polyvinylidene difluoride membrane (Thermo Fisher Scientific). Western blotting with anti-HA and anti-MYC antibody was performed as previously described (62 (link)) with the following modifications. The primary antibodies were diluted 1:1,000 (anti-HA, H6908; Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween (TBST) or 1:1,000 (anti-MYC, SAB4301136; Sigma-Aldrich) in 5% skim milk TBST and incubated at 24°C for 2 h. Membranes were imaged by using a ChemiDoc Touch MP Imaging System (Bio-Rad, USA).

Anti-HERC3 (HPA039170, 1:500 dilution), anti-HA (SAB4300603, 1:5000 dilution), and anti-Myc (SAB4301136, 1:2000 dilution) were purchased from Sigma-Aldrich. Anti-EIF5A2 (16386-1-AP, 1:500 dilution), anti-E-Cadherin (20874-1-AP, 1:5000 dilution), N-Cadherin (22018-1-AP, 1:2000 dilution), Vimentin (10366-1-AP, 1:2000 dilution), anti-GAPDH (60004-1-Ig, 1:2000 dilution), anti-Flag (20543-1-AP, 1:5000 dilution), and anti-GST (HRP-66001, 1:5000 dilution) were purchased from Proteintech. Anti-TGF beta1 (ab215715, 1:1000 dilution), antiphospho-Smad2 (phosphor S467) (ab280888, 1:1000 dilution), antiphospho-Smad3 (phosphor S423 + S425) (ab52903, 1:1000 dilution), anti-Smad2 (ab40855, 1:1000 dilution), and anti-Smad3 (ab40854, 1:1000 dilution) were purchased from abcam. The antibodies were used according to the corresponding instructions.