Anti actin c4

Whole cell protein extracts from U2OS cells overexpressing ectopic PTEN and mutations were prepared by cell lysis in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% IGEPAL CO-630 (Nonidet P-40), 2 mM Na₃VO₄, 100 mM NaF, 1mM PMSF, 1 μg/ml of aprotinine, 20 mM Na₄P₂O₇), followed by centrifugation at 15200 g for 10 min and collection of the supernatant. Yeast cell extracts were obtained by standard procedures. Proteins (50–100 μg in the case of mammalian cells; 20–50 μg in the case of yeast) were resolved in 10% SDS-PAGE under reducing conditions and transferred to PVDF membranes. Immunoblot was performed using anti-phospho-Ser473-Akt + anti-phospho-Thr308-Akt and anti-Akt antibodies (Cell Signaling Technologies), anti-PTEN 425A mAb (Andrés-Pons et al., 2005) or rabbit polyclonal anti-PTEN antibodies (Upstate), anti-GAPDH (Santa Cruz Technology) or anti-actin C4 (MP Biomedicals, France) antibodies, followed by horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse (Calbiochem) antibodies. For determination of phospho-Akt content, bands were quantified using ImageQuantTL software (Amersham Biosciences).

Total protein extraction was performed using dry mycelia from cultures grown in minimal media supplemented with NaNO₃ at 25 °C, as previously described [44 (link)]. Total proteins (25–50 μg, estimated by Bradford assays) were separated in 8–10 % (w/v) polyacrylamide gels. Immunodetection was performed on PVDF membranes (GE Healthcare Life Sciences, Amersham, United Kingdom) using primary anti-FLAG M2 (Sigma-Aldrich, St. Louis, MO, USA), anti-GFP (Roche Diagnostics, Basel, Switzerland), anti-actin (C4) (MP Biomedicals, Santa Ana, CA, USA) and an HRP-linked secondary antibody (Cell Signaling Technology Inc, Danvers, MA, USA). Blots were developed using the LumiSensor Chemiluminescent HRP Substrate kit (Genscript, Piscataway, NJ, USA) and SuperRX Fuji medical X-ray films (FujiFILM, Minato City, Tokyo, Japan).