Chop d46f1

Soluble human His-tagged recombinant TRAIL was generated in our laboratory as described^{44 (link)}. Anti-caspase 8 antibody was generously provided by Dr. Gerald Cohen (Leicester University, UK). Antibodies against p-AKT^Ser473, AKT, p-P70S6K, P70S6K, PERK (C33E10), eIF2α (D7D3), p-eIF2α and CHOP (D46F1) were obtained from Cell Signaling Technology (CA, USA). Antibodies against ATF4, GAPDH, and GCN2 were obtained from Santa Cruz Technology (Santa Cruz, CA, USA). Anti-TRAIL-R2 antibody was purchased from R&D Systems (Minneapolis, USA). Anti-FLIP (7F10) antibody was from Enzo Life Sciencies (Farmingdale, NY, USA). p4E-BP1 and 4E-BP1 antibodies were from Upstate Millipore (NY, USA). FITC-conjugated secondary antibodies were from DAKO (Cambridge, UK). Anti-TRAIL-R1-PE and anti-TRAIL-R2-PE monoclonal antibodies for surface receptor analysis were from Biolegend (San Diego, CA, USA). Aminooxyacetate, non-essential amino-acid solution (100×), cycloheximide, dimethyl 2-oxoglutarate (α-KG), and l-Asparaginase from E.Coli were from Sigma-Aldrich (St. Louis, MO, USA). Torin1 was purchased from TOCRIS Bioscience (Bristol, UK). Rapamycin was purchased from LC laboratories (Woburn, MA, USA). Epigallocatechin Gallate (EGCG) was purchased from Selleck Chemicals (Houston, Texas, USA).

Cells were harvested in RIPA cell lysis buffer (Thermo Fisher, 89900) and heated for 5 min at 100 °C. Equal quantities of denatured protein samples were resolved on 10% SDS-polyacrylamide gels and were then transferred onto nitrocellulose membranes (Roche, Basel, Switzerland). After blocking with 5% non-fat dry milk in TBS/0.05% Tween-20, the membranes were incubated with a specific primary antibody (1:1000) followed by a horseradish peroxidase-conjugated secondary antibody. Rabbit antibodies against XBP1s (clone D2C1F, #12782), CHOP (D46F1, #5554), ATF4 (D4B8, #11815), IRE1α (14C10, #3294), p-eIF2 (D9G8, #3398), and GAPDH (14C10, #2118) were purchased from Cell Signaling Technologies. Proteins were visualized using ECL reagents (Pierce, Rockford, IL, USA) and analyzed with Fiji software to quantify intensity of the bands. Protein levels were normalized to GAPDH. Expression of XBP1s in the untreated samples is below the limit of detection. For XBP1s quantification, we used overexposed film to quantify the fold difference between untreated samples and the 0.5 μg/mL Tm control. The XBP1s levels from other treatments are then interpolated based on their intensities in comparison with the 0.5 μg/mL sample. In addition, we have used serial dilution to obtain the levels of expected band intensity to verify the quantification is in the right range.