On 2015 Sep 21, Toni Mueller commented:

Please note regarding the subcellular fractionation protocol to obtain a synapse-enriched fraction (SYN) as indicated in the Methods and Figure 1: subsequent experiments in our lab have indicated that it is not necessary to combine P1 and P2 prior to triton solubilization. While combining these pellets enhanced the protein yield of the SYN fraction and the triton-insoluble other heavy/intermediate membrane fraction to facilitate western blot sample preparation, the SYN fraction generated by triton solubilization of only P2 appears to have less contamination by nuclear proteins. The steps performed in calculating GABA(A) receptor subunit expression and ratios do account for this potential confound (subunit expression is normalized to an inhibitory synapse marker-gephyrin, a loading control- VCP, and subunit expression in total homogenate), but other researchers and labs measuring proteins with both nuclear and synaptic expression patterns should be aware of this limitation in the fractionation method.

On 2015 Sep 21, Toni Mueller commented:

Please note regarding the subcellular fractionation protocol to obtain a synapse-enriched fraction (SYN) as indicated in the Methods and Figure 1: subsequent experiments in our lab have indicated that it is not necessary to combine P1 and P2 prior to triton solubilization. While combining these pellets enhanced the protein yield of the SYN fraction and the triton-insoluble other heavy/intermediate membrane fraction to facilitate western blot sample preparation, the SYN fraction generated by triton solubilization of only P2 appears to have less contamination by nuclear proteins. The steps performed in calculating GABA(A) receptor subunit expression and ratios do account for this potential confound (subunit expression is normalized to an inhibitory synapse marker-gephyrin, a loading control- VCP, and subunit expression in total homogenate), but other researchers and labs measuring proteins with both nuclear and synaptic expression patterns should be aware of this limitation in the fractionation method.