Phosphatase inhibitor cocktails 3

To compare protein expression in IVD NP tissues by Western blotting, proteins were extracted from IVD NP tissues. Harvested tissues were homogenized using the MS-100R bead-beating disrupter (Tomy Seiko, Tokyo, Japan) for 30 s twice at 4 °C in the T-PER tissue protein extraction reagent (78510; Thermo Fisher Scientific, Waltham, MA, USA) with protease and phosphatase inhibitors (Protease inhibitor cocktail [25955-11; Nacalai Tesque], Phosphatase inhibitor cocktails 2 [P5726; Sigma-Aldrich], and Phosphatase inhibitor cocktails 3 [P0044; Sigma-Aldrich]). Finally, soluble proteins were collected by centrifugation at 20,000× g for 15 min at 4 °C, and the protein concentration was determined using a bicinchoninic acid assay (23227; Thermo Fisher Scientific). Samples were stored at −80 °C.

Vegetative hyphae of PH-1 were harvested from 12 h YEPD cultures and treated with 500 μM 2,4-D or 0.4% ethanol (v/v) for 30 min. Total proteins were then isolated with protein lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich, USA), phosphatase inhibitor cocktails 2 (Sigma-Aldrich, USA) and phosphatase inhibitor cocktails 3 (Sigma-Aldrich, USA) and separated on 10% PAGE gels as described (Zhang et al. 2018 (link)). Western blots were detected with the anti-TpGY phosphorylation-specific (for detection of phosphorylated FgHog1 with the TGY dual phosphorylation site), anti-FgHog1, anti-TpEY phosphorylation-specific (for detection of phosphorylated Gpmk1 and Mgv1 MAP kinases with the TEY dual phosphorylation site), and anti-ERK2 antibodies (Cell Signaling Technology, USA) as described (Zhang et al. 2018 (link), 2017 ) for assaying the phosphorylation and expression levels of FgHog1, Gpmk1, and Mgv1 MAP kinases. Detection with an anti-β-tubulin (anti-tub2) antibody was used as the protein loading control as described (Wang et al. 2019 ).