MIP/ZEO, MIP/SP, or HCT116 cells were seeded in 60 -mm dishes. After 48 h, cells were treated with 5-FU, CPT-11 or tunicamycin at indicated concentration and time periods. Cells were extracted in lysis buffer [1% Triton-X 100, 120 mM NaCl, 50 mM Tris-HCl, pH 7.5] supplemented with 1% proteinase inhibitor cocktail (Sigma) followed by protein quantification with Bradford assay (Bio-Rad). Equal amount of proteins were separated by SDS-PAGE under reducing conditions and electrotransferred onto a PVDF membrane (Millipore). Blots were incubated with primary antibodies in TBST (TBS containing 0.1% Tween-20) overnight at 4 °C. [1:1000 anti-GRP78 (sc-13968, Santa Cruz and cat. 3177 Cell Signaling Technology (CST)); 1:1000 anti-SPARC (AON-5031, Haematologic Technologies Inc.); 1:200 anti-pPERK (cat. 649402, BioLegend); 1:1000 anti-PERK (CST); 1:1000 anti-peIF2α (cat. 3597, CST); 1:1000 anti-eIF2α (sc-11386, Santa Cruz); 1:250 anti-ATF4 (sc-200, Santa Cruz); 1:1000 anti-pIRE1α (ab124945, Abcam); 1:1000 anti-IRE1α (cat. 3294, CST); 1:10,000 anti-β-actin (G043, ABM); 1:1000 anti-calnexin (cat. 2679, CST) and 1:2000 anti-DNA-PK (cat. 4602, CST)]. The blots were then incubated with anti-mouse immunoglobulin (IgG)-HRP (SH023, ABM) or anti-rabbit IgG-HRP (cat. 7074, CST) followed by enhanced chemiluminescence (ECL) detection. For immunoprecipitation analysis, conformation-specific secondary antibodies were used [1: 5000 anti-mouse IgG-HRP (Rockland) and 1:5000 anti-rabbit IgG-HRP (Rockland)].
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