Ab32371

The treated MDA-MB-468 cells were lysed with the cell lysis buffer. Protein concentration was measure by the BCA Protein Assay kit (Beyotime Biotechnolgy, Beijing). Lysates were separated in 10% SDS-PAGE and then transferred onto the nitrocellulose membrane (Millopore, USA). The membrane was blocked with 5% skimmed milk in TBST buffer at room temperature for 1 h. After that, the membranes were incubated with anti-Caspase 3 (1:5000, ab32351, Abcam, Cambridge, MA, USA), anti-Cleaved Caspase 3 (1:500, ab32042, Abcam), anti-Ki67 (1:5000, ab92742, Abcam), anti-PCNA (1:2000, ab92552, Abcam), anti-Bak (1:10000, ab32371, Abcam), anti-Bax (1:1000; ab32503, Abcam), anti-Bcl-2 (1:1000, ab32124, Abcam), anti-apaf-1 (1:1000, ab2001, Abcam), anti-Cytochrome c (1:5000; ab133504, Abcam), anti-Akt (1:1000; #4685, Cell Signaling Technology, Danvers, MA), anti-phosphorylated-Akt (1:1000, #4060 CST), anti-p38 (1:1000, #8690, CST), anti-phosphorylated p38 (1:1000, #4511, CST), anti-NF-κB (1:1000, #8242, CST), anti-p21 (1:1000; ab109520, Abcam), anti-p27 (1:5000; ab32034, Abcam) and GAPDH (1:2500, ab9485, Abcam) at 4°C overnight and then secondary antibodies (1:5000) were incubated for 1 h at room temperature. The proteins were visualized by using the enhanced chemiluminescence reagent (Bio-Rad, USA).

Total proteins were isolated from cells using RIPA lysis buffer. After loading 25 μg cellular protein samples on SDS‐PAGE gels, proteins of the same weight from each group were separated and transferred to PVDF membranes. Subsequently, membranes were blocked with 5% skimmed milk for 1 h at room temperature and later incubated with primary antibodies overnight at 4°C. The main antibodies include rabbit anti‐human CD1E antibody (1: 1000, ab187157, Abcam), rabbit anti‐human STK11 antibody (1: 1000, ab138386, Abcam), rabbit anti‐human Bax antibody (1: 1000, ab32503, Abcam), rabbit anti‐human Bak antibody (1: 10000, ab32371, Abcam), rabbit anti‐human Bcl‐2 antibody (1: 1000, ab32124, Abcam), rabbit anti‐human AMPK antibody (1: 1000, ab207442, Abcam), rabbit anti‐human p‐AMPK antibody (1: 1000, ab133448, Abcam), and rabbit anti‐human β‐actin antibody (ab115777, Abcam). Following incubation with primary antibodies, they were incubated with secondary antibody IgG (1: 2000, ab97051, Abcam) for 1 h at room temperature. Protein bands were detected with enhanced chemiluminescence reagents (Merck Millipore). β‐actin was used as an internal reference.

SHP099 (HY-100388A) and bortezomib (HY-10227) were purchased from MedChemExpress (Princeton, NJ, United States). RMC-4550 (S8718) was obtained from Selleckchem (Houston, TX, United States). The primary antibodies against SHP2 (#3397), phospho-SHP-2 (Tyr542) (#3751), and P21 (#2947) were obtained from Cell Signaling Technology. Anti-GAPDH (10494-1-AP) was purchased from Proteintech. The primary antibodies against ERK (AF1051) and phospho-ERK (AF1891) were procured from Beyotime Biotechnology. The antibodies specific for BAK (ab32371) and cleaved caspase-3 (ab32042) were obtained from Abcam. HRP-conjugated secondary antibodies were purchased from Servicebio.

The whole cell lysates (Beyotime, Shanghai, China) were used to harvest total protein. The equal amount of protein extract was then separated by SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride membrane. Subsequently, membranes were blocked in 5% non-fat dry milk. The membranes were incubated with the appropriate dilutions of primary antibodies against NOD1 antibody (1:1000, ab217798, Abcam, Cambridge, MA, US), BAK1 (1:1000, ab32371, Abcam, Cambridge, MA, US), NLRP6 (1:1000, ab58705, Abcam, Cambridge, MA, US), and GAPDH (1;1500; Abcam, Cambridge, MA, USA) antibody sampler kit (9782 T) at 4 °C. The more detailed steps have been illustrated as previously described [24 (link)].