Protease inhibitor cocktails 2 and 3

Cells were transfected 48 h prior to cell collection, treated with 20 μM MG132 for the last 8 h, and lysed in lysis buffer [50 mM Tris.HCl (pH7.4), 150 mM NaCl, 10% Glycerol, 1 mM EDTA], 1% Triton-X-100, phosphatase inhibitors and protease inhibitor cocktails 2 and 3 (Sigma-Aldrich). The cell lysates were cleared by centrifugation and incubated with Flag M2 protein A/G (Cell Signaling) beads overnight. The beads were boiled after extensive washing and the proteins were detected by Western blotting, followed by quantification using Image J software (http://rsb.info.nih.gov/ij). For endogenous immunoprecipitation, SOX9 was immunoprecipitated from lysates that were prepared from two 10-cm dishes with a rabbit polyclonal antibody against SOX9 (Millipore, AB5535), using Protein A/G plus-Agarose IP Reagent (Santa Cruz), following the manufacturer's instructions. For immunoprecipitation under denatured conditions, cells were lysed in denaturing buffer (1% SDS, 5 mM EDTA, phosphatase and protease inhibitors), boiled for 5 min and passed through a 26-g needle several times. Lysates were diluted 1:10 in the non-denaturing lysis buffer as used for Western blotting. Immunoprecipitation was carried out as described above.

Following 48-h treatments, the supernatants from the SH-SY5Y cells were collected into sterile 15 mL Falcon tubes. They were centrifuged (Sigma3-16K, Sigma-Aldrich, UK) for 5 min at 3000 rpm to remove cell debris, and stored at –80°C. The remaining adhered cells in the flasks were washed twice with 5 mL phosphate buffered saline (PBS), detached, and suspended in PBS. Once detached, the cells were transferred into 15 mL sterile, Falcon tubes and centrifuged at 1000 rpm for 10 min. After draining off excess PBS, the cell pellet was lysed in 250μL volume of lysis buffer (RIPA buffer, pH 8.0: containing 50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.5% Sodium deoxycholate, 0.5% (v/v) NP-40, 1% sodium dodecyl sulphate. 1/100 final of phenylmethanesulphonyl or PMSF and 5 mM dithiothreitol, 5% protease inhibitor cocktails 2 and 3 (Sigma-Aldrich, UK). The cells were vortex mixed and incubated on ice for 10 min with further vortex mixing in between. The cell homogenate now in Eppendorf tubes were centrifuged at 14,000 rpm for 20 min (Sigma 1–14 microfuge). The liquid phase was withdrawn, transferred into new pre-labelled 1.5 mL Eppendorf tubes, and used to determine total protein following a protein assay (see below). All cell lysates were stored at –80°C until needed for western blotting (AβPP cleavage products).