Analysis of MAPK phosphorylation via western blot

To examine the impact of Msg5 on MAPK phosphorylation, mycelia from CFLH16, mutant, and complementation strains were cultured in a liquid shaking CM medium at 28°C and 180 rpm for a duration of 24 hours. Mycelium samples were collected before (time 0) and 30 minutes post the addition of 5 mm H₂O₂. Total protein was extracted using RIPA lysis buffer and further treated with a phosphatase inhibitor cocktail (100×), protease inhibitor cocktail (100×, EDTA-free), and Phenyl methane sulphonyl fluoride (PMSF), subsequently placed on ice for 30 minutes with stirring at 10-minute intervals. The Western blot procedure was carried out as detailed by Nordzieke et al. [72]. Briefly, protein samples were run on 7.5% SDS-polyacrylamide gels (Omni-Easy™One-Step PAGE Gel Fast Preparation Kit, PG211) and transferred onto a nitrocellulose membrane using the wet transfer method. Phosphorylation of Mpk1 and Fmk1 MAPKs was detected with the Phospho-ERK1/2 rabbit mAb antibody ((Thr202/Tyr204)/(Thr185/Tyr187); Zen bio; cat#R24245) [64]. Beta tubulin (6D8) mouse mAb antibody (Zen bio; cat#250007) served as a loading control. The hybridized bands were visualized using the ECL hypersensitive chemiluminescence solution (Zen bio, cat#17045) in a Tanon automatic chemiluminescence imager. Densitometric quantification of western blot signals was carried out using the ImageJ software, as described [72]. The ratio of phospho-MAPK to Beta tubulin signal was determined and normalized to the wildtype level under non-inducing conditions (time 0) for each individual blot. The experiment was repeated thrice. Statistical analyses were conducted using the t-test for unequal variances, also known as Welch’s test.