Total protein in Tris-buffered saline (TBS) and neutralized formic acid (FA) extracts was quantified using the Ready to Use Bradford Reagent (Bio-Rad) and for TBS-Triton X100 (TBSX) extracts the BCA Protein Assay Kit (Pierce).
For western blot analysis of TBS-X fractions protein (20 μg) was separated on 4–12 % Bis-Tris gels (Invitrogen) and transferred onto low-fluorescence PVDF membranes. Membranes were blocked (1 hr. room temperature) with 5 % non-fat milk in TBS containing 0.1 % Tween 20 (TBST), then incubated (4 °C, overnight) with primary antibodies against post synaptic density protein (PSD-95, 1:1000, Cell Signaling), synaptophysin (1:1000, Cell Signaling), the EGF receptor (1:100, Santa Cruz Biotechnology) or actin (1 hr. room temperature, 1: 20,000, Cell Signaling) in TBST with 5 % bovine serum albumin (BSA). Membranes were incubated in secondary antibodies (Jackson Immunoresearch) with TBST wash (3X5min) in between steps and imaged using an Odyssey ® Fc Imaging System. Image J was utilized to quantify actin normalized proteins.
For western blot analysis of TBS-X fractions protein (20 μg) was separated on 4–12 % Bis-Tris gels (Invitrogen) and transferred onto low-fluorescence PVDF membranes. Membranes were blocked (1 hr. room temperature) with 5 % non-fat milk in TBS containing 0.1 % Tween 20 (TBST), then incubated (4 °C, overnight) with primary antibodies against post synaptic density protein (PSD-95, 1:1000, Cell Signaling), synaptophysin (1:1000, Cell Signaling), the EGF receptor (1:100, Santa Cruz Biotechnology) or actin (1 hr. room temperature, 1: 20,000, Cell Signaling) in TBST with 5 % bovine serum albumin (BSA). Membranes were incubated in secondary antibodies (Jackson Immunoresearch) with TBST wash (3X5min) in between steps and imaged using an Odyssey ® Fc Imaging System. Image J was utilized to quantify actin normalized proteins.