Anti mpk1 antibody

Western blot analysis of MAPK phosphorylation levels was performed as described [36 (link),38 (link),39 (link)]. Phosphorylated Mpk1 and Fmk1 was detected using rabbit antiPhospho-p44/42 MAPK (Erk1/2) antibody (Thr202/Tyr204; Cell Signaling Technology; #4370), while total Mpk1 and Fmk1 protein was detected with mouse monoclonal anti-Mpk1 antibody (Santa Cruz Biotechnology; sc165979) and a custom-designed polyclonal anti-Fus3 antibody [36 (link)]. Mouse anti-α-tubulin antibody (Sigma-Aldrich; #T9026) was used as the loading control.

Fresh mycelia (200 mg) of each strain were finely ground and suspended in 1 ml of extraction buffer (50 mM Tris-HCl, pH7.5, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, 2 mM PMSF) and 10 μl of protease inhibitor cocktail (Sangon Co., Shanghai, China). After homogenization with a vortex shaker, the lysate was centrifuged at 10 000 g in a microcentrifuge for 20 min at 4°C. The resulting proteins were separated on 10% denaturating polyacrylamide gel (SDS-PAGE) and transferred to Immobilon-P transfer membrane (Millipore, Billerica, MA, USA) with a Bio-Rad electroblotting apparatus. The monoclonal anti-GFP ab32146 (Abcam, Cambridge, MA, USA) antibody was used at a 1:5000 to 1:10000 dilution for BcMkk1-GFP immunoblot assays. The phosphorylated BcBmp1 and BcBmp3 were detected with phospho-p44/42 MAPK antibody (Cell Signaling Technology, Boston, MA, USA). The total BcBmp1 and BcBmp3 were detected using p44/42 MAPK antibody (Cell Signaling Technology Inc., Beverly, MA, USA) and anti-Mpk1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively. Incubation with a secondary antibody and chemiluminescent detection were performed as described previously [92 (link)]. The experiment was conducted three times independently.