Rabbit anti rpl7a

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.

Western blot analysis was performed as described^{75 (link)}. In brief, protein samples were transferred on a nitrocellulose membrane and blocked in 2%(w/v) BSA. Proteins were detected using rabbit anti-Rck 1:1000 (MBL), goat anti-DDX6 1:1,000 (Abnova), mouse anti-ß-actin 1:5000 (Sigma-Aldrich), rabbit anti-RPL7A 1:1000 (Abcam), mouse anti-Gephyrin 1:1000 (Synaptic Systems) and mouse anti-GFP 1:500 (self-made, kind gift by Angelika Noegel, Köln) primary antibodies and donkey anti-rabbit IRDye 680RD conjugated 1:10,000, donkey anti-mouse IRDye 800CW conjugated 1:10,000 and donkey anti-goat IRDye 680RD conjugated 1:10,000 (LI-COR) secondary antibodies. Primary antibody binding was detected using the LI-COR Odyssey IR scanner.

For protein analysis, cells were lysed in hot 3× SDS loading buffer. Nucleic acids were digested using Benzonase. Proteins were separated with an SDS-PAGE and transferred to nitrocellulose membrane (pore size 0.2 μm). Primary antibodies (anti-eEF2, anti-p-eEF2, anti-phospho(p)-mTOR, anti-Caspase3, and anti-PARP1 [1:1.000 dilution, all from rabbit, Cell Signaling Technology; # 2332, #2331, #2971, #9662, and #9542]; mouse anti-Rps6, rabbit anti-p-Rps6 [1:1.000 dilution, Cell Signaling Technology; #2317 and #4858]; mouse anti-β-III Tubulin [1:10.000 dilution, Sigma-Aldrich; #T8578]; mouse anti-ACTB [1:5.000 dilution, Sigma-Aldrich; #A2228]; rabbit anti-Rpl7a [1:1.000 dilution, Abcam; #ab70753]; rat anti-HA [1:100 dilution, Helmholtz Center Munich Antibody Core Facility, clone 3F10], mouse anti-PMY [1:5.000 dilution, Sigma-Aldrich; #MABE343]) were diluted in bovine serum albumin blocking solution and incubated with membranes overnight. Membranes were washed and incubated with IRdye labeled secondary anti-mouse, anti-rabbit or anti-rat antibodies (1:10.000 dilution, LI-COR Biosciences). Fluorescence signals were detected with an Odysee scanner (LI-COR Biosciences).