Biotinylated phalloidin

F-actin was isolated from cerebral cortices (E17.5) or neuronal cell cultures (E14+5DIV) after modification of a published protocol 37 (link). Four cortices for each condition were dissected and transferred into 1 ml of homogenization buffer (100 mM Na₂HPO4 – 100 mM NaH₂PO4 at pH 7.2, 2 mM ATP, 2 mM MgCl₂, 0.1% Triton X-100) containing phosphatase and protease inhibitor cocktails (Sigma). For analysis on cultures, 16 wells of 24-well plates were pooled for each condition. When needed, cells were incubated for 10 min with 10 μM of the calcium ionophore A23187 (Tocris) before the lysis. Neurons were then lysed and homogenized, like tissues, at 4°C into 1 ml of homogenization buffer by pipetting up and down using a 18G needle. Biotinylated phalloidin (1 unit for each condition, Invitrogen) was then added to homogenates and incubated under rotation for 1 h at room temperature. In parallel, streptavidin-coated magnetic beads (50μl for each condition; Invitrogen) were blocked for 30 min in PBS (Sigma) with 5% bovine serum albumine (Sigma), washed and added to homogenates with biotinylated phallodin for 1 h at room temperature. The precipitated material was then washed five times with homogenization buffer, resuspended in 1X laemmli sample buffer (Bio-Rad) and analyzed by western blotting using primary antibodies against actin and CaMKIIβ according to the protocol described below.