Cell lysates or protein samples were resolved on 7% SDS-PAGE gels and transferred to PVDF using a wet transfer apparatus and blocked in 5% milk solution in PBS-Tween20. The membranes were probed with appropriate primary and secondary antibodies (see below) and the bands were visualized using ECL Western blotting substrates (Thermo Fisher Scientific or GE Healthcare). Where appropriate, the membrane was stripped with Restore Western Blot Stripping Buffer (ThermoFisher) and re-probed. Membranes were incubated with the following primary antibodies: anti-PICK1 (Neuromab) and anti-GFP (Neuromab).
Secondary antibodies conjugated to HRP were obtained from GE Healthcare and used at 1:10,000 dilutions. For densitometry, Western blot films were scanned and analyzed using ImageJ followed by the appropriate statistical analysis carried out using GraphPad Prism. All error bars on graphs represent the standard error of the mean.
Secondary antibodies conjugated to HRP were obtained from GE Healthcare and used at 1:10,000 dilutions. For densitometry, Western blot films were scanned and analyzed using ImageJ followed by the appropriate statistical analysis carried out using GraphPad Prism. All error bars on graphs represent the standard error of the mean.