G box scanner

Human Arp2/3 complex (crosslinking mutant, 0.25 μM) was incubated with 1.5 μM WCA, 5 μM Cort_1-76, or 1.5 μM WCA + 5 μM Cort_1-76 for 30 min at RT in ATP-supplemented KMEI buffer. Freshly prepared BMOE (ThermoFisher Scientific) in dimethyl sulfoxide (DMSO) was added to a final concentration of 16 μM at 21 °C. Reactions were quenched at the indicated time points (Fig. 2) with the addition of an equal volume of 2x SDS-loading buffer (LI-COR Biosciences) supplemented with 100 mM β-mercaptoethanol. Samples were loaded onto 12% SDS-PAGE gels, transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad) and immunoblotted with anti-Arp3 antibody (1:5000 dilution, Santa Cruz Biotechnology, sc-48344). Membranes were imaged using a G:BOX scanner (Syngene). Densitometric analysis was performed using Image Lab 6.1 (Bio-Rad). Mean and SD values were calculated from three independent experiments.

Cells were lysed on ice in lysis buffer (15 mM PBS, 2% NP-40, 0.2% SDS, 10 mM EDTA, 1% protease inhibitor cocktail, 1% phosphatase inhibitor cocktail). After centrifugation, the supernatant was collected and protein concentration evaluated by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Lysates containing 10 to 20 μg of proteins were re-suspended in Laemmli buffer, then proteins were resolved on 10% acrylamide SDS-polyacrylamide gel electrophoresis and then electro-transferred to nitrocellulose membranes for Western blot (WB) analysis. Protein transfer was evaluated by red Ponceau S staining (Sigma-Aldrich, St Louis, CA, USA). The membranes were blocked in a 5% milk solution in TBS (0.1% Tween 20) and incubated 12 hours at 4°C with primary antibodies. The reactivity was revealed by incubation (1 hour at 20°C) with HRP-conjugated secondary rabbit anti-goat IgG followed by chemiluminescence reaction performed with electrochemiluminescence (ECL) detection reagents (GE Healthcare, Little Chalfont, UK) and film exposure. The WB bands reactivities were quantified by densitometry analysis using a G-Box scanner and the associated GeneSys software (Syngene, Cambridge, UK). The films were scanned and the bands optical density was measured with GeneTools software (Syngene, Cambridge, UK). Expression of α-tubulin was used as a loading control.