Total s6

LNCaP95 cells were serum-starved for 24hr, followed by treatment with DMSO, EPI-002 (25uM), enzalutamide (10 uM), BEZ235 (15nM) or a combination for 1hr prior to addition of R1881 or EtOH for 48hr. Antibodies used were: AR (1:1000; Santa Cruz), AR-V7 (1:400; Precision), p110α (1:500; BD Bioscience), p110β (1:1000; abcam), p100γ (1:1000; abcam), p110δ (1:1000; abcam), UBE2C (Boston Biochem; 1:1000), PTEN (1:1000), pS6 (1:2000), pAktThr308 (1:1000), pAktSer473 (1:2000), p4EBP1 (1:1000), total-Akt (1:1000), total-S6 (1:1000), total-4EBP1 (1:1000), pERK/MAPK (1:1000), total-ERK/MAPK (1:1000) from Cell Signaling technology (Danvers, MA). β-actin (1:10,000, Abcam) was used as a loading control.

Cells were collected and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.5% NP-40; 50 mM NaF with protease inhibitors) and incubated on ice for 30 min. Lysates were centrifuged at 20,817g at 4 °C for 10 min and supernatant was collected. Protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein samples were mixed with SDS Laemmli loading buffer, boiled and electrophoresed using NuPAGE Bis-Tris Gels (Life Technologies), then transferred onto PVDF membranes (Millipore). Blocking was performed for 45 min using TBST supplemented with 5% non-fat dry milk and blotting performed with primary antibodies at 4 °C for 16 h. The following antibodies were used: ADSL (Abcam #ab154182), GMPS (Cell Signaling #14602), PRPS1 (Abcam #ab154721), MYC (N-262, Santa Cruz #sc-764), total AKT (Cell Signaling #4691), phospho-AKT (S473, Cell Signaling #9271), total S6 (Cell Signaling #2317), phospho-S6 (235/236, Cell Signaling #4858), phospho-S6 (240/242, Cell Signaling #5364), total p70 S6K (Cell Signaling #2708), phospho-p70 S6K (Cell Signaling #9234) and α-tubulin (Sigma #T6074). All antibody validation information is available in the product’s manual. For western blotting, the dilution was 1:500 for MYC antibody and 1:1,000 for all other antibodies.