C digit chemiluminescence scanner

Western blot was performed according to the method described previously [29 (link)] to assess Keap-1/Nrf-2/HO-1 and p38 MAPK/NF-κB pathways’ protein expression. Briefly, renal tissue samples were mixed with RIPA buffer containing protease inhibitor; the extracted protein was measured using a Nano Drop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Thereafter, 50 μg of the total extracted protein was separated via SDS-PAGE and blotted onto PVDF membranes. PVDF membranes were blocked by incubation in TBS enclosing 3% bovine serum albumin and 0.1% Tween 20 for one hour at room temperature. PVDF membranes were washed (TBS containing 0.1% Tween 20) and incubated first with a 1:1000 dilution of the primary antibodies (Keap-1, Nrf2, HO-1, P38 MAPK and NF-κB) for two hours and, then, with a 1:5000 dilution of the secondary antibody at room temperature. Keap-1 (sc-514914), Nrf2 (sc-722), HO-1 (sc-136960), P38 MAPK (sc-7973), NF-κB p65 (sc-8008), reference gene (β-actin; SC-130656) and goat anti-rabbit immunoglobulin (Ig) G-horseradish peroxidase (HRP) (sc-2030) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The chemiluminescence produced was detected with the C-DiGit chemiluminescence scanner (LI-COR, Lincoln, NE, USA), and the band intensity was analyzed using the scanner software.

Western blot was performed according to the method described previously [30 (link)]. Briefly, cytoplasmic and nuclear proteins were extracted from the kidney tissue using the Protein Extraction Kit (KeyGen Biotech Co. Ltd., Nanjing, China), containing protease inhibitor and phosphatase inhibitor cocktails (Sigma Aldrich, Darmstadt, Germany), according to the manufacturer’s protocols. The total protein extracted concentration was considered using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Afterward, 50 µg of the total extracted protein was separated via sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and blotted onto PVDF membranes. PVDF membranes were blocked by incubation in Tris-buffered saline (TBS), containing 3% bovine serum albumin and 0.1% Tween 20 for 1 h at room temperature. After washing with TBS containing 0.1% Tween 20, the membranes were incubated firstly with the primary antibodies (1:300 dilution) for 2 h, and then goat antirabbit HRP-conjugated (as secondary antibody; at a 1:5.000 dilution) at room temperature. The chemiluminescence produced from the luminol reagent was detected with the C-DiGit chemiluminescence scanner (LI-COR, Lincoln, NE, USA), and the band intensity was analyzed using the scanner software.

Mycelial colonies were grown for 24 h at 27C on M2 plates topped with cellophane. For each plate (~30 colonies), the harvested mycelium was recovered on 500 µL of extraction buffer (25 mM Tris-HCl pH7.5, 150 mM NaCl, 0.5% NP40, 1 mM PMSF, 1 mM DTT, 1 × SigmaFast Protease Inhibitor) and added with 167 µL of 50% trichloroacetic acid. After 60 min at −75C, the samples were centrifugated (16,900 × g, 15 min), and the pellet was washed twice with 1 mL of cold 80% acetone by centrifugation (16,900 × g, 5 min), and air-dried. The proteins were recovered from the pellet with 100 µL of 0.1 NaOH, 1% sodium dodecyl sulfate (SDS), and 20µL of 6 × SDS loading buffer. Samples were boiled for 5 min, and 8 µL of each sample was loaded per well for an SDS-polyacrylamide gel electrophoresis. For Western blot analyses, GFP was detected with a monoclonal mouse anti-GFP antibody (B2, Santa Cruz Biotechnology, SC-9996) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) with a rabbit anti-GAPDH antibody (GeneTex, GTX100118), using HRP-conjugated anti-mouse (Jackson ImmunoResearch, 115–035-003) or anti-rabbit (Invitrogen, 31460) secondary antibodies, and the SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific). Images were captured with a C-DiGit Chemiluminescence Scanner (LI-COR), and the intensity of the protein bands was quantified on ImageJ.