Anti fmrp blg 834601

Protein extraction was performed in ice-cold RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF; CST-8553S, Cell Signaling Technology) and 1% protease inhibitor cocktail (p2714, Sigma-Aldrich). Cell lysates were incubated for 20 min on ice, centrifuged, and the supernatants were separated on 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to nitrocellulose membranes (0.2 μm; PB7320, Thermo Fisher Scientific) using BIO-RAD Mini Trans-Blot Cell. After electrotransfer, the blots were blocked with PBST containing 5% BSA and incubated 1 h at RT with primary antibodies anti-FMRP (BLG-834601, Biolegend) and anti-β-actin (ab8226, Abcam). Blots were then washed with PBST and incubated 1 h at RT with the secondary antibody anti-mouse horseradish peroxidase (CST-7076, Cell Signaling Technology). Blots were detected by enhanced chemiluminescence Western blotting substrate EZ-ECL (RPN2106, Biological Industries) and developed by MYECL Imager (Thermo Fisher Scientific).

Cells were fixed with 4% paraformaldehyde (PFA; P6148, Sigma-Aldrich) for 20 min at room temperature (RT). Blocking was performed with blocking solution: 10% Fetal Bovine Serum (FBS) or 5% Goat serum (GS) with 0.2% Triton X 100 in PBS for permeabilization. Cells were then incubated with primary antibodies (anti-SSEA4, CST-4755, Cell Signaling Technology, Danvers, MA, USA; anti-TRA-1-60, ab16288, Abcam, Cambridge, UK; anti-OCT4, sc-5279, Santa-Cruz, Starr County, TX, USA; anti-FMRP, BLG-834601, Biolegend, San Diego, CA, USA; anti-Tuj1, BLG-801201, Biolegend; anti-PAX6, BLG-901301, Biolegend; anti-MAP2, sc-20172, Santa Cruz; anti-SYN1, AB1543, Merck, Darmstadt, Germany; anti-PSD-95, MAB1596, Merck) diluted in blocking solution for 1 h at RT, washed 3 times with PBS, and incubated with secondary antibodies (donkey anti-mouse Alexa Fluor 488, A21202, Thermo Fisher Scientific, Waltham, MA, USA; goat anti-rabbit Alexa Fluor 594, A11012, Thermo Fisher) for another 1 h at RT in the dark, and counterstained with DAPI for nucleus localization (D1306, Thermo Fisher Scientific). Cells were mounted with Fluoromount aqueous mounting medium (00-4958-02, Thermo Fisher Scientific). Bright-field, phase, and fluorescence images of cells were obtained using an Olympus IX51 inverted light microscope (Olympus, Tokyo, Japan).