Amersham enhanced chemiluminescence detection reagents

Western blot analyses of VeA and LaeA were performed as described in Jeong and Yu (44 (link)). Briefly, 2-day-old conidia (2 × 10⁸), vegetatively grown cells (6, 12, 24, 48 h), asexually developing cells (12, 24, 48 h), and sexually developing cells (12, 24, 48 h) were collected and resuspended in the lysis buffer containing a 1× protease inhibitor cocktail. Then, samples were homogenized by a mini-beadbeater with 0.5 mm zirconia/silica beads. Protein concentrations were measured by the Bio-Rad Protein Assay (Bio-Rad). Approximately 15 μg of total proteins per lane were separated on 4 to 12% gradient SDS-PAGE (Bio-Rad) gel and transferred onto immobilon-P PVDF membrane (Millipore). The membrane was blocked with a blocking buffer, incubated with the monoclonal anti-FLAG antibody produced in mice (clone M2, Sigma-Aldrich), and then incubated with HRP-conjugated anti-mouse IgG (Millipore). The membrane was developed using Amersham enhanced chemiluminescence detection reagents (GE Healthcare).

Cells were harvested and lysed in lysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 1% nonidet P-40, 10 mM EDTA, 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 10 mg/mL leupeptin). After centrifugation at 13,000 rpm for 15 min, protein concentration in each supernatant was determined using the Bio-Rad protein assay reagent (Bio-Rad, Richmond, CA, USA). For western blotting, the same amount of proteins was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Bio-Rad). After blocking with 5% skim milk in PBS at 4 °C, the blots were probed with primary antibodies overnight. After incubation with secondary antibodies for 1 h, bands of interest were visualized with Amersham enhanced chemiluminescence detection reagents (GE healthcare, Piscataway, NJ, USA). Representative images from at least two independent experiments are shown. The relative band intensity was quantified by ImageJ 1.52 and was normalized to β-actin.

The cell lysates were prepared in a lysis buffer (20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 10 mM EDTA, 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride and 10 mg/ml leupeptin). After centrifugation at 13,000 rpm for 15 min, the protein concentration in each lysate was determined using Bio-Rad protein assay reagent (Bio-Rad, Richmond, CA, USA) according to the manufacturer’s protocol. Equal amounts of proteins were loaded and separated by SDS-PAGE. After transfer to nitrocellulose membranes (Bio-Rad), blots were blocked overnight with 5% skim milk in PBS at 4°C and probed with primary antibody overnight. Protein bands were detected with Amersham enhanced chemiluminescence detection reagents (GE healthcare, Piscataway, NJ, USA).