Ab218123

This study utilized RIPA buffer (Beyotime Institute of Biotechnology) that contained protease K inhibitor for extracting total proteins, which were then separated through 12% SDS-PAGE, followed by transfer onto PVDF membrane. Subsequently, membrane was immersed within 5% defatted milk under ambient temperature for a 2 h, followed by overnight incubation under 4 °C using corresponding primary antibodies: EZH2 (1:500, ab191080; Abcam, Cambridge, MA, USA), p-STAT3 (1:2000, ab76315; Abcam, Cambridge, MA, USA), STAT3 (1:1000, ab68153; Abcam, Cambridge, MA, USA), Mcl-1 (1:1000, ab32087; Abcam, Cambridge, MA, USA) and p-Bcl-2 (1:1000, ab218123; Abcam, Cambridge, MA, USA), H3K27me3 (1:1000, ab6002; Abcam, Cambridge, MA, USA) with GAPDH being the loading reference. Then, the membrane was subject to secondary antibody incubation (1:5000, ab96899; Abcam, Cambridge, MA, USA). Protein band visualization was performed using Chemidoc EQ system (Bio-Rad Laboratories, Inc.).

Treated HUVECs or tissues were lysed by RIPA lysates (P0013B, Beyotime, Beijing). After being processed by the ultrasonic cell disruptor, the sample was centrifuged at 12,000°g at 4°C for 5°min on the ice. The supernatant was collected in a new EP tube. The BCA protein assay kit (#23227; Thermo, United States) was utilized to evaluate the protein concentration. The protein sample was separated by SDS-PAGE gel electrophoresis and transferred to the PVDF membrane. The membrane was sealed with 5% skimmed milk powder (232100-500°g, BD, United States) at room temperature for 2°h. Following being incubated with the primary antibodies against PI3K (1:1,000; ab32089; Abcam, United States), p-PI3K (1:1,000; ab86714; ab32089), Akt (4298; CST, United States), p-Akt (12694), mTOR (2972; Cell Signaling, United States), p-mTOR (2971; Cell Signaling), LC3-I / II (1:1,000; ab62721; Abcam), Beclin-1 (1:1,000; ab210498; Abcam), P62 (1:1,000; ab211324; Abcam), Bcl-2 (1:1,000; ab218123; Abcam), Bax (1:1,000; ab3191; Abcam), Caspase-3 (1:1,000; ab179517; Abcam) and β-actin (1:1,000; ab115777; Abcam) overnight at 4°C, samples were incubated with horseradish Peroxidase (HRP) labeled goat anti-rabbit (ZB-2301; ZSGB-BIO, Beijing, China) or anti-mouse (ZB-2305; ZSGB-BIO) IgG at room temperature for 2°h. The expression of target proteins was quantified by ImageJ software.

LNCaP and 22RV1 cells were treated with RIPA buffer (Beyotime) to obtain proteins, which were quantified using BCA (Pierce, USA). A 10% sulfate-polyacrylamide gel electrophoresis was utilized to separate the equivalent amount of protein, which was then delivered to a polyvinylidene fluoride membrane. The membrane was sealed with PBST containing 5% skim milk, and then coexisted with CDT1 (ab202067; Abcam, UK), Bax (ab3191; Abcam), Bcl-2 (ab218123; Abcam), CyclinD1 (ab134175; Abcam), CDK4 (ab68266; Abcam), or GAPDH antibody (ab181602; Abcam) at 4°C overnight. The membrane was incubated with the secondary antibody (ab205718; Abcam) for 1 h at 37°C. Protein bands were observed using enhanced chemiluminescence (ECL) substrates (Pierce, USA) [28 (link)].