Phospho tuberin tsc2

Histologic evaluation was done by preparing 4-μm transverse sections from 4% paraformaldehyde-fixed, paraffin-embedded tissue. Hematoxylin and eosin (H&E) staining was performed according to the standard protocol [19] (link). IHC and ISH experiments were carried out as previously described [20] . Primary antibodies used in the experiments were rabbit anti-IL6 (1:200), rabbit anti-caspase 3 (1:100), mouse anti-proliferating cell nuclear antigen (PCNA) (1:2000), and rabbit anti-JAK1 (1:200) from Abcam (Cambridge, MA). Mouse anti-phospho-PI3K (1:100) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit antibodies for phospho-Tuberin/TSC2 (1:200), phospho-mTOR (1:200), phospho-4EBP1 (1:200), phospho-RS6K (1:200), and phospho-STAT3 (1:200) were purchased from Cell Signaling (Danvers, MA). For ISH experiment, partial cDNA sequences were PCR-amplified using the appropriate primers (refer to Supplementary Table 2) and cloned into pCRII vector (Invitrogen). Riboprobes were generated using T7 or SP6 digoxigenin labeling kit (Roche Diagnostics GmbH, Mannheim, Germany). Hybridization was done at 65°C overnight, and a series of stringent wash was done at 68°C. Hybridized riboprobes were detected by anti-dig antibody binding and visualized by incubating with an NBT/BCIP AP substrate solution (Roche Diagnostics GmbH). Counterstaining was done with neutral red.

Antibodies to Akt, phospho-Akt(S473), phospho-Akt(T308), phospho-Tuberin/TSC2(T1462), Tuberin/TSC2, GSK-3β, phospho-GSK-3β(S9), mTOR, Raptor, phospho-4E-BP1(T37/46), p70S6K, phospho-p70S6K(T389), LC3A/B, ULK1, phospho-ULK1(S757) and Anti-Rabbit IgG Fab2 Alexa Fluor (R) 488 conjugate were obtained from Cell Signaling Technology (Beverly, MA, USA). β-Actin and HA-probe antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Piperlongumine was obtained from Indofine Chemical Company (Hillsborough, NJ, USA). Chloroquine diphosphate, Puromycin dihydrochloride, Hexadimethrine bromide and N-Acetyl-L-Cysteine were obtained from Sigma-Aldrich (St Louis, MO, USA). Bafilomycine A1 was obtained from LC Laboratories (Woburn, MA, USA).