Anti hsp70

Shikonin (SK) was purchased from Tokyo Chemical Industry (Tokyo, Japan), and doxorubicin (Dox) was from Sigma (St. Louis, MO, USA). The three antibodies used for depletion of specific DAMP proteins in tumor cell lysate (TCL) were anti-HSP70 (rabbit plyoclonal; GeneTex), anti-CRT (rabbit polyclonal; Abcam) and anti-HMGB1 (rabbit plyoclonal; GeneTex). The same antibodies and anti-β-actin antibody (rabbit polyclonal; Abcam) were also used as primary antibodies for western blot analyses. HRP-conjugated secondary antibody (goat polyclonal; Abcam) was used as a secondary antibody.

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).