Anti phospho s6 ribosomal protein ser235 236 antibody

Protein extracts were obtained using trichloroacetic acid (TCA) extraction as described previously (Motamedi et al., 2008 (link)). Briefly, cell pellets, prepared from 5 mL of logarithmically growing cells (32°C in EMM + aa medium, OD₆₀₀ ∼ 0.7–1) frozen in liquid nitrogen, were resuspended in 0.5 mL of cold TCA buffer (20mM Tris (pH 8.0); 50 mM ammonium acetate; 2 mM EDTA; 2 mM PMSF; 2 mM Leupeptin; and 2 mM aprotinin), 0.5 mL of cold 30% TCA, and 0.5 mL of cold glass beads. Pellets were lysed in MagNA Lyser (Roche) instrument (3 × 30 s at 6000rpm with a 3 min on ice incubation in between). After lysis, the extracts were centrifuged at 20,000 rcf for 5 min and the resulting pellets were washed once in acetone. Acetone was removed and the protein pellets were resuspended in 40 μL TRIS buffer (0.1 M Tris-HCl pH 8.0.1 mM EDTA, 1%SDS) and 10 μL 5X loading buffer (containing 1 mM DTT and 1 mM PMSF) and boiled at 100°C for 5 min. Protein extracts were separated by SDS-PAGE (12%), transferred onto a nitrocellulose filter and analyzed by Western blot using anti-Phospho-S6 Ribosomal protein (Ser235/236) antibody (Cell Signaling, 4858). Tubulin was used as the protein loading control (Sigma, T6074). Immunoblots were developed using the enhanced chemiluminescence procedure.

This study was approved by the Human Research Ethics Committee at the Royal Children's Hospital, Melbourne (ID 29077). Written informed consent was obtained from the participant's parents.
The patient had drug‐resistant focal epilepsy and underwent three epilepsy surgeries. Information was obtained from medical records, scalp and intracranial EEG recordings, PET and MRI scans, operative records and photographs, and histopathology.
Resected brain tissue was snap frozen or stored as formalin‐fixed paraffin‐embedded (FFPE) tissue at the time of surgery. Genomic DNA preparation, HaloPlex^HS targeted panel sequencing, droplet digital PCR (ddPCR) and immunohistochemistry were performed as previously described.⁴ The assay ID of the Custom Taqman SNP Genotyping Assay for MTOR NM_004958.4:c.4375G > C was AN2XHET (Thermo Fisher Scientific) and immunostaining utilized anti‐Phospho‐S6 Ribosomal Protein (Ser235/236) antibody (Cell‐Signalling, #2211, #4858).