Western Blot Analysis of NMD Pathway Proteins

Proteins were electrophoresed in 6–14% polyacrylamide, transferred to a nitrocellulose (Bio-Rad) or polyvinylidene difluoride (Millipore) membrane and probed as described^{5 (link)} using the following antibodies (see Supplementary Table 6): anti-p-UPF1 S1116 (1:1,000), anti-UPF1 (1:2,000), anti-FMRP (1:2,000), anti-SMG7 (1:2,000), anti-eIF4A3 (1:1,000), anti-PABPC1 (1:2,000), anti-β-actin (1:5,000), anti-CBP80 (1:2,000), anti-eIF4E (1:2,00), anti-SMG5 (1:2,000), anti-SMG6 (1:1,000), anti-UPF2 (1:2,000), anti-UPF3X/UPF3B (1:2,000), anti-SMG1 (1:2,000), anti-mTOR (1:2,000), anti-HERC2 (1:2,000), anti-GADD45B (1:2,000), anti-ATF3 (1:2,000), anti-ARHGEF18/p114RhoGEF (1:2,000), anti-GAPDH (1:5,000), anti-OCT4 (1:2,000), anti-SOX2 (1:2,000), anti-β3-tubulin/TUJ1 (1:2,000), anti-MAP2 (1:2,000), anti-BRN2/POU3F2 (1:2,000), anti-FOXG1 (1:2,000), anti-doublecortin/DCX (1:2,000), anti-synapsin1/SYN1 (1:2,000), anti-calnexin (1:5,000), anti-MS2CP (1:2,000), anti-GFP (1:500), anti-HA HRP (1:1,000) or anti-FLAG HRP (1:1,000; Sigma–Aldrich). Western blots were quantitated using Image Studio Lite Version 4.0 (LI-COR Biosciences). Dilution standards assured that quantitations were in the linear range of analysis.