Membrane protein extraction kit

The membrane and mitochondrial protein were extracted immediately after the rats' hearts were isolated, according to the operating instructions of Membrane Protein Extraction Kit (Boster, Wuhan, China) and Mitochondria Isolation Kit (Boster) respectively. The total proteins were extracted from the frozen ventricle tissues by RIPA. Total 40 μg samples were fractionated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA) as previously described [1 (link)], and then done with primary antibodies against Cx43, Bcl-2, Bcl-xL, Bax, AKT, phosphorylation of AKT (p-AKT), active Caspase-3, Beclin-1, LC3-II (1:1000, rabbit species, all from Cell Signal Technology) and survivin (1:1000, goat species, Santa Cruz Biotechnology, SantaCruz, CA, USA) at 4°C overnight. Rinsed three times in TBS, the filters blots were incubated with HRP-conjugated rabbit or goat second antibodies (1:5000; Kang Chen Biotechnology, Guangzhou, China) for 1 hr at room temperature. The bands were visualized by an ECL Western-blotting Detection Reagents (Catalogue RPN2106; GE Healthcare, Fairfield, CT, USA) with LAS-3000 detect system. To quantify the immunobloted bands, optic density was analysed using ImagePro 5.0 (Media Cybernetics Inc., Silver Spring, MD, USA).

To determine ALA-PDT induced HSP70, HMGB1, and CRT expression, membrane proteins of ALA-PDT-treated PECA tumor cells were extracted with a membrane protein Extraction Kit (BOSTER, China). PECA cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Tris base 50 mM, NaCl 150 mM, NP40 1%, sodium deoxycholate 0.25%, EDTA 1 mM) containing a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The protein concentration was measured using a BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of proteins were separated by SDS-polyacrylamidegel electrophoresis (PAGE) on 10% gels and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% (w/v) non-fat milk in TBS [Tris-buf-fered saline (pH 7.4)] containing 0.1% (v/v) Tween 20 (TBST) and then incubated with primary antibodies (anti-HSP70, anti-HMGB1 and anti-CRT antibody) overnight at 4°C. The membranes were then washed three times (15 min each) with TBST, and incubated with an alkaline phosphatase-conjugated secondary antibody (1:2000) for 1 h at room temperature. The color reaction was developed using NBT (p-nitro-blue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3-indolyl-phosphate; Sigma).