Mouse monoclonal anti rack1

Immunofluorescence staining on neurons and SH-SY5Y cells was performed as previously described [24 (link)]. Briefly, after two rinses with Phosphate Saline Buffer (Na₂HPO₄ 10mM, KH₂PO₄ 1.8mM, NaCl 137mM, KCl 2.7mM, pH 7.4), SH-SY5Y and embryonic primary cells were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature, then rinsed again with PBS and permeabilized with PBS-Triton-X 0.5% for 10 min at room temperature. After blocking in PBS-BSA 2% at 37 °C for 30 min, cells were incubated for 30 min at 37 °C with the rabbit monoclonal anti-FMRP (Abcam, 1:100) and mouse monoclonal anti-RACK1 (BD Biosciences, 1:200) primary antibodies (dissolved in blocking solution). After washes with PBS, the following secondary antibodies were incubated for 30 min at 37 °C in the dark: goat anti-mouse, goat anti-rabbit (Alexa Fluor^® secondary antibodies, Molecular Probes), dissolved 1:500 in PBS-DAPI. After mounting with 80% glycerol, cells were visualized by fluorescence microscopy (Axio Scope, Zeiss, Viterbo, Italy).

Immunoblotting was performed on protein extracts obtained with lysis buffer as previously seen for histidine pull-down. Equal amounts of proteins were loaded on a 10% SDS-PAGE and transferred to the PVDF membrane (Millipore). After blocking in 5% Bovine Serum Albumin in PBS1X for 30 min at 37 °C, blots were incubated with the following primary antibodies: mouse monoclonal anti-RACK1 (BD Biosciences, 1:2000), mouse monoclonal anti-β-actin (Sigma, 1:1000), mouse monoclonal anti-puromycin (Millipore, 1:10,000), mouse monoclonal anti-Myc 9B11 (Cell Signaling, 1:1000), rabbit monoclonal anti-FMRP (Abcam, 1:1000), rabbit polyclonal anti-phospho-FMRP (Abcam, 1:500), rabbit monoclonal anti-phospho-ERK (Cell Signaling, 1:1000), rabbit monoclonal anti-phospho-rpS6 (Cell Signaling, 1:1000). Secondary HRP-conjugated anti-mouse or anti-rabbit antibodies and ECL reagent (1:5000, GE Healthcare) were used. For RACK1, mouse HRP-conjugated anti-IgM (1:5000, Sigma) was used.