Lysates were separated by SDS-PAGE. Proteins were transferred to nitrocellulose (pore size 0.2 µm). Membranes were blocked in blocking solution (2% (w/v) BSA, 0.1 vol% Tween 20, 0.1% (w/v) sodium azide in 1× TBS pH 7.5) for at least 1 h. Primary antibodies were diluted in blocking buffer and incubated overnight at 4 °C. The following antibodies were used in this study: polyclonal antibodies: rabbit anti-Pum2 (Abcam) 1:10,000, rabbit anti-Btz (self-made, [52 (link)]) 1:500, rabbit anti-Rpl7a (Abcam) 1:1000, rabbit anti-Rps6 1:1000, rabbit anti-phospho-Rps6 1:1000, rabbit anti-PABP1 (all Cell Signaling) 1:1000, goat anti-Vinculin (Santa Cruz) 1:200; monoclonal antibodies: mouse anti-eIF4E (BD) 1:1000, mouse anti-eIF2s1 (Cell Signaling) 1:1000, mouse anti-FMRP (gift from Utz Fischer, Würzburg) 1:1000, mouse anti-β-III-Tubulin (Sigma Aldrich) 1:10,000, and rabbit anti-phospho-mTOR (Cell Signaling) 1:1000).
Membranes were washed in PBS supplemented with 0.2 vol% Tween 20. Primary antibodies were detected using infrared dye labeled secondary anti-rabbit, anti-goat, or anti-mouse antibodies (all 1:10,000, Li-COR Biosciences). Membranes were scanned using the Li-Cor Odysey IR scanner.
Membranes were washed in PBS supplemented with 0.2 vol% Tween 20. Primary antibodies were detected using infrared dye labeled secondary anti-rabbit, anti-goat, or anti-mouse antibodies (all 1:10,000, Li-COR Biosciences). Membranes were scanned using the Li-Cor Odysey IR scanner.