For each peptide slope factors were determined by

linear regression fits to the measured PVs versus dilution factor of the load reflecting Onalespib ic50 each peptide’s specific MS signal intensity ( Figure S2; loads of the three concatenated standards were normalized to each other by their abundancenorm values; Zolles et al., 2009). These slope factors were then used to normalize the respective peptide PVs in APs or BN-PAGE slices to an equimolar basis. The relative molar abundance of each protein was then calculated as intensity-weighted mean (AP data sets) or median (BN-PAGE samples) of the respective normalized peptide PVs. To establish protein profiles across BN-PAGE samples ( Figure 2), the respective PV tables were preprocessed: (1) see more each individual slice measurement (i.e., LC-MS data set) was scaled by dividing its average PV to a sliding average PV of the neighboring two slices (window of 5) to account for variations in slice thickness, peptide recovery and LC-MS sensitivity and (2) a filter was applied to eliminate false-positive PV assignments (identified as solitary values without backup from the neighboring two slices or > 10-fold outliers with respect to the average of corresponding PVs in the neighboring two slices) and to bridge gaps resulting from false-negative assignments

(individual missing values Resminostat were replaced by the corresponding PV average of the neighboring slices, if available). The resulting relative molar protein abundances were finally smoothened by averaging (window

of 3). Hippocampal sections of adult Wistar rats (CA3 area, 80 nm thick) were processed for the postembedding immunogold labeling as described earlier (Kulik et al., 2002 and Schwenk et al., 2009) and stained with affinity-purified mouse anti-GluA2/4 (Millipore, #MAB396) and two different rabbit anti-GSG1-l ABs (raised against aa 83-102 and aa 257-278; Figure S5B). Secondary ABs (1:20; British Biocell International, Cardiff, UK) were coupled to either 10 nm gold particles (for GluR2/4) or 15 nm gold particles (for GSG1-l). Electrophysiological recordings from giant outside-out patches excised from oocytes were performed at room temperature (22°C –24°C) as described previously (Berkefeld et al., 2006). Currents were recorded with an EPC9 amplifier, low-pass filtered at 3 kHz and sampled at 5–10 kHz. Pipettes made from thick-walled borosilicate glass had resistances of 0.4–0.8 MΩ when filled with intracellular solution (Kint; in mM) 120 KCl, 5 HEPES, 10 EGTA, pH adjusted to 7.2. Extracellular solution (Kext) applied to outside-out patches was composed as follows (mM): 120 KCl, 5 HEPES, 1.3 CaCl2 (pH 7.2).