Tuberin tsc2

Cultured fibroblasts were lysed in 20 mM Tris, pH7.5, 150 mM NaCl, 20 mM NaF, 2.5 mM Na₄P₂O₇ × 10H₂O, 1 mM β-glycerophosphate, 1% NP-40, 1 mM benzamidine, 10 mM 4-nitrophenyl phosphate, 0.1 mM PMSF (reagents from Sigma). Ten percent SDS-PAGE was run using 5 ug of total protein lysate. Proteins were transferred to Invitrolon PVDF (Life Technologies). For blocking membranes, TBS/0.1% Tween 20/5% NF milk for 1 hr was used. Antibodies were diluted in blocking buffer and incubated overnight at 4°C. Antibodies which reacted with tuberin/Tsc2, p-S6 ribosomal protein (ser235/236), and total S6 ribosomal protein were purchased from Cell Signaling (#4308, #2211 and 2217, respectively). Monoclonal anti-β-actin was purchased from Sigma (#A5441).

Cholesterol, POPC, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) were purchased from Avanti Polar Lipids. Human Wnt3a protein was purchased from R&D Systems (Cat no. 5036-WN-010; lot no. RSK9221052). siRNA for human TSC2 was purchased from Integrated DNA Technologies (Ref. no. 311363879) and the transfection reagent JetPRIME was from Polyplus-transfection. Antibodies against β-catenin (Cat no. 9587S), active (nonphospho) β-catenin (Cat no. 8814S), Tuberin/TSC2 (Cat no. 4308S), p70 S6 kinase (S6K) (Cat no. 2708S), phospho-p70 S6K (pS6K) (Cat no. 9205S), and glyceraldehyde 3-phosphate dehydrogenase (Cat no. 5174S) were purchase from Cell Signaling Technologies. Anti-Rabbit IgG, HRP-linked antibody (Cat no. 7074S) and anti-Mouse IgG as well as HRP-linked antibody (Cat no. 7076S) were also from Cell Signaling Technologies. HEK293T and HeLa cells were purchased from ATCC. Chemicals for Cholesterol oxidase assay activity, including 4-aminoantipyrine (Cat no. A4382-10G), phenol (Cat no. P1037-25G), and peroxidase from horseradish (Cat no. P8375-1KU) were purchased from Millipore Sigma.