Bca protein kit

The harvested CSCs were lysed with RIPA Lysis Buffer (Aspen, Canada, USA) containing the protease inhibitor cocktail (Roche, Basel, Switzerland), followed by centrifugation at 12000 rpm at 4℃ for 5 min. Protein concentration was determined by BCA protein kit (Aspen, Canada, USA). Total protein was electrophoresed on 10% SDS-PAGE and then transferred to PVDF membrane. Subsequently, the membrane was incubated with the primary antibodies overnight at 4℃, followed by the corresponding horseradish peroxidase conjugated secondary antibodies at room temperature for 30 min. GAPDH served as the internal control. Protein bands were captured by enhanced chemiluminescence (ECL) substrate solution kit (Aspen, Canada, USA) for 1 min and analyzed with AlphaEaseFC software. Primary antibodies used in this study are listed in Table 2.

After the same treatment as described in 2.10, HSFs were harvested and lyzed in RIPA buffer (AS1004, ASPEN) containing inhibitors of protease and phosphatase (AS1008, ASPEN), followed by centrifugation (12000 rpm × 5min). The BCA protein kit (AS1086, ASPEN) was used to assess the protein contents in the lysate. The cell lysate samples (50μg/lane) were separated by SDS-PAGE gels (AS1012, ASPEN) and transferred to PVDF membranes (IPVH00010, Millipore). After being blocked with 5 % bovine serum, the membranes were incubated with primary antibodies at 4 °C overnight. The membrane was then incubated with the secondary antibody at room temperature for 1 h after being washed with TBST. The proteins were detected using an ECL chemiluminescence kit (AS1059, ASPEN). The primary antibodies included p-p38 (28796-1-AP, Proteintech), p38 (14064-1-AP, Proteintech), p-JNK(#4668, CST), JNK(#9252, CST), p-ERK (28733-1-AP, Proteintech), ERK (11257-1-AP, Proteintech), Bax (#2772, CST), Cleaved caspase3 (19677-1-AP, Proteintech), HIF-1α (20960-1-AP, Proteintech), p53 (10442-1-AP, Proteintech), Beclin-1 (AF5128, affinity), LC3-II (ab48394, abcam), β-Actin (TDY051, 1:10000).