, 2007), anti-TARP γ-8 (Frontier Institute, RB Af1000-1), OTX015 anti-TARP γ-2 (Upstate, #07-577), anti-CKAMP44 (kind gift of Dr. R. Sprengel, von Engelhardt et al., 2010), anti-GSG1-like

(raised in rabbit against aa 257-278 of Swiss-Prot accession Q6UXU4, affinity purified), anti-PRRT1 (raised in rabbit against aa 36-54 of Swiss-Prot accession Q6MG82, affinity purified), anti-Noelin1 (R&D Systems, #AF4636), and anti-FLAG (Sigma, #F3165). After brief washing with the respective detergent buffer bound proteins were eluted with Laemmli buffer (DTT added after elution). Isolated proteins were shortly run into SDS-PAGE gels, silver stained, cut in two pieces of MW > 50 and MW < 50 kDa, and in-gel digested with trypsin ( Pandey and Mann, 2000). Western analyses were performed with anti-GluA1-a, anti-GluA2 (Millipore, MAB397), anti-GluA2/3, anti-GluA3, anti-GluA4-a, anti-TARP γ-2, anti-TARP γ-8, anti-CKAMP44, anti-GSG1-like (Sigma, #HPA014479), and anti-CNIH-2/3 ABs. The AB-stained bands were

www.selleckchem.com/products/NVP-AUY922.html visualized by anti-mouse, -rabbit, -goat IgG-HRP (all Santa Cruz), and ECL+ (GE Healthcare). Two to six consistent peptides specific for each of the identified AMPAR constituents (Table S4) as well as three control proteins were selected and randomly fused in silico to form three N- and C-terminally tagged standard (QconCAT) proteins (84, 60, and 82 peptides resulting in QconCAT proteins

of 907 aa, 743 aa, and 942 aa, respectively). The corresponding gene sequences were synthesized (GenScript) and subcloned in a modified pET16 vector; calibration proteins were expressed in Escherichia coli BL21(DE3). After verification of full-length expression by dual western blots using anti-tag ABs, two-fold dilutions of the QconCAT proteins (seven to nine steps) were separated by SDS-PAGE. The corresponding protein bands were visualized by Coomassie staining, excised, and separately digested by from trypsin for subsequent triplicate mass spectrometric analysis. Extracted postdigest peptide mixtures dissolved in 0.5% (v/v) trifluoroacetic acid were analyzed by nano-LC-MS/MS with a LTQ FT Ultra mass spectrometer as described (Müller et al., 2010). Precursor signals were acquired with a target value of 1,000,000 and a nominal resolution of 100,000 (FWHM) at m/z 400 (scan range 370 to 1700 m/z). Up to five data-dependent CID fragment spectra per scan cycle were acquired in the ion trap with a target value of 10,000 (maximum injection time 400 ms) with dynamic exclusion enabled.