α actin

Western blot was performed as previously described by our group (31 (link)) using the following primary antibodies: VIM (rabbit polyclonal, dilution 1:3,000, catalog number 10366-1-AP, ProteinTech, Manchester, UK), CDH1 (mouse monoclonal, Clone 36, dilution 1:500, catalog number 610182, BD Transduction Laboratories, San Jose, CA, USA) and α-actin (mouse monoclonal, Clone C4, dilution 1:1,000, catalog number 691001, MP Biomedicals, Solon, OH, USA). Antibody binding was revealed by incubation with anti-mouse (rabbit polyclonal, dilution 1:5,000, catalog number A9044, Sigma-Aldrich, Merk Merck Life Science, Madrid, Spain) or anti-rabbit (goat polyclonal, dilution 1:5,000, catalog number sc-2004, Santa Cruz Biotechnology, Dallas, TX, USA) IgG peroxidase conjugate secondary antibodies. Protein expression was assessed using HPR Chemiluminescent Substrate Reagent Kit (catalog number WP20005, Invitrogen, Thermo Fisher Scientific) and ChemiDoc (BIO-RAD, Hercules, CA, USA).

Protein extracts for immunodetection were obtained through the alkaline lysis protocol [40 (link)]. Briefly, approximately 6 mg of lyophilized mycelium was resuspended in 1 ml lysis buffer (0.2M NaOH, 0.2 % β-mercaptoethanol). After trichloroacetic acid (TCA) precipitation, 100 µL Tris-base (1 M) and 200 µl of loading buffer [62.5 mM Tris/HCl pH=6.8, 2 % SDS (p/v), 5 % β-mercaptoethanol (v/v), 6 M urea and 0.05 % bromophenol blue (p/v)] were added. Samples were then loaded and proteins separated on SDS-polyacrylamide gels (%10), and electro-transferred to nitrocellulose filters (Trans Blot Turbo transfer packs) using the Trans Blot Turbo system (BioRad) following the manufacturer’s instructions. HA₃-tagged proteins were detected using a monoclonal rat α-HA3 (1 : 1000; clone 3F10; Sigma-Aldrich). Actin, used as loading control, was detected using monoclonal mouse α-actin (1 : 5000; clone C4; MP Biomedicals). Peroxidase-conjugated goat anti-rat IgG +IgM (1 : 4000; Southern Biotech) or anti-mouse (1 : 4000; Jackson ImmunoResearch Laboratories) were used as secondary antibodies. Western blots were developed using the Amersham Biosciences ECL kit and chemiluminescence was detected using a Chemidoc image system driven by Image Lab Touch software (version 2.2.0.08; BioRad). Images were processed to a minimum using Image Lab software (version 6.0; BioRad).