Ab128995

On Day 7, 10⁵–10⁷ differentiated cells were collected into 2 mL centrifuge tubes, gently mixed with 2 mL Dulbecco’s phosphate-buffered saline (DPBS), and centrifuged at 1,000 × g for 5 min. After washing the cell pellet twice with DPBS, 4% paraformaldehyde was immediately added to fix the cells for 10 min, and this was followed by centrifugation at 1,000 × g for 5 min. The pellet was washed three times with DPBS, and 0.4% Triton X-100 was added for 15 min before centrifuging the cells at 1,000 × g for 5 min. The pellet was washed three times with DPBS, and 5% bovine serum albumin (BSA)-DPBS was added. After centrifugation at 1000 × g for 5 min, primary antibodies against RPS6 (ab80158, Abcam, Cambridge, UK) and RPS3 (ab128995, Abcam) were separately added to the pelleted cells, and they were allowed to stand at 25°C for 2 h. After washing three times with DPBS, a fluorescent-labeled goat anti-rabbit secondary antibody was added, incubated at room temperature for 1 h, and washed three times with DPBS. The cells were then spread evenly and dried on clean glass slides. DAPI was then added, anti-fluorescence quencher coverslips were mounted, and images were captured using an upright fluorescence microscope (LSM710, Zeiss, Germany).