Tumor cell lysate samples were prepared as previously described [8 (link), 66 (link)]. To assay for expression of ICD-associated markers, 4T1 TCL protein samples were resolved by SDS PAGE using 8, 10 or 15% stepwise gels. The resolved proteins were transferred onto a PVDF membrane (Novex, San Diego, CA) and blotted with anti-hnRNPA1 (rabbit polyclonal; GeneTex), anti-HMGB1 (rabbit plyoclonal; GeneTex), anti-HSP70 (rabbit plyoclonal; GeneTex), anti-CRT (rabbit polyclonal; Abcam), or anti-β-actin (rabbit polyclonal; Abcam). The membrane was blocked with 5% non-fat dry milk in PBST buffer [phosphate-buffered saline (PBS) containing 0.1% Tween 20] for 60 min at room temperature. Blotted membranes were then incubated overnight at 4°C with specific, commercially available antibodies (1:1,000 dilutions). Loading of equal amounts of protein was assessed using mouse β-actin protein as a reference. The blots were rinsed three times with PBST buffer for 5 min each. Washed blots were incubated with HRP-conjugated secondary antibody (goat polyclonal; Abcam; 1:100,000 dilution) and washed again three times with PBST buffer. The transferred proteins were visualized with an enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia Biotech, Buckinghamshire).
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