Anti phospho s6 ser235 236

Samples were electrophoresed on 11% SDS polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). The membranes were incubated in 5% bovine serum albumin in Tris-buffered saline containing Tween-20 (50 mM Tris-HCl pH 7.4, 150 mM NaCl, and 0.03% Tween-20) and then incubated with primary antibodies anti-pan-AKT, anti-phospho-AKT (Ser473), anti-phospho-AKT (Thr308), anti-pan-S6, anti-phospho-S6 (Ser235/236) (Cell Signaling Technologies, Danvers, MA, USA), and anti-α-tubulin (Sigma-Aldrich), followed by incubation with a horseradish peroxidase-conjugated secondary antibody. Immunoreactive proteins were visualized with Western Lightning Plus-ECL (PerkinElmer, MA, USA). Densitometric measurement of bands on western blotting was performed using ImageJ software (US National Institutes of Health, Bethesda, MD, USA).

For immunofluorescence analysis, NPCs were plated on chamber slides (Lab-Tek) or differentiated into neurons on poly-L-ornithine/laminin-coated glass-bottom culture dishes (MatTek). Cells were fixed in 4% (vol/vol) paraformaldehyde for 15 min followed by blocking in phosphate-buffered saline (PBS) containing 8% fetal bovine serum (vol/vol) for 1 h. The primary antibodies were prepared in PBS solution with 2 mg/ml saponin and incubated for 2 h at room temperature or overnight at 4°C. The cells were then washed in PBS and incubated with the corresponding fluorochrome-conjugated secondary antibodies for 1 h. Vectashield mounting medium plus DAPI (Vector Laboratories) was applied to label the nuclei. The following primary antibodies were used: anti-mTOR (Cell Signaling Technology, 2972) 1:100; anti-phospho-mTOR-Ser2448 (Cell Signaling Technology, 5536) 1:100; anti-S6 Ribosomal Protein (Cell Signaling Technology, 2217) 1: 200; anti-phosphoS6-Ser235/236 (Cell Signaling Technology, 2211) 1:100; anti-phospho-4EBP1-Thr37/46 (Cell Signaling Technology, 2855) 1:100; anti-LAMP1 (Developmental Studies Hybridoma Bank, H4A3) 1:200; anti-p62 (BD Biosciences, 610832) 1:100; anti-Tuj1 (Neuromics, MO15013) 1:200; anti-TFEB (MyBioSource, MBS855552) 1:50. The secondary antibodies used were Alexa fluor 488- or 594-conjugated mouse or rabbit (Life Technologies), both at a 1:200 or 1:400 dilution.

For immunofluorescence staining, specimens were fixed in PBS with 4% formaldehyde, washed twice with PBS, and immersed in 0.1% Triton X-100 overnight at 4 °C, followed by washing 3 times with PBS. The following primary antibodies were incubated overnight at 4 °C: anti-phospho-mTOR (Ser2448) (1:200, Bioworld), anti-cleaved caspase-3 (1:200, Epitomics), anti-fibronectin (1:200, Santa Cruz) anti-phospho-ULK1 (ser757) (1:1000, Cell Signaling), anti-phospho-S6 (Ser235/236) (1:200, Cell Signaling) or anti-LC3A (1:50, Cell Signaling). After incubation with primary antibodies, cells were washed with PBS and then incubated with fluorescence-conjugated secondary antibodies (1:5000, FITC, Jackson ImmunoResearch, Westgrove, PA) for 2 hr at room temperature. After nuclear staining with 40,6-diamidino-2-phenylindole (DAPI, D9542, Sigma-Aldrich), spheres were then placed in a Lab-Tek^®II chamber (Nunc, Naperville, IL) and analyzed with a confocal fluorescent microscope (Olympus FV10i). Negative controls without utilizing primary antibodies were also prepared to rule out nonspecific labeling.