Ab32503

Total protein of cells in each group was isolated using protein lysis buffer and Bradford method (Thermo Fisher Scientific, Waltham, MA, USA) was applied for protein quantitation. Protein of 50 μg was firstly subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to Polyvinylidene Fluoride membrane (Millipore, Billerica, MA, USA). Then 5% skimmed milk power was added at 37°C to terminate the reaction for 1 hour. After that, primary antibodies of mouse anti human CNE1 (ab3927, 1:1000), Bcl2 (ab32124, 1:1000), Bax (ab32503, 1:1000), Caspase3 (ab32503, 1:1000), Cleaved‐Caspase3 (ab32042, 1:100), β‐actin (1:1000, Abcam) (all purchased from Abcam, Cambridge, MA, USA) were added at 4°C for overnight. The membrane was washed with PBST for three times, each for 5 minutes before secondary antibodies of rabbit anti mouse which were labeled by horse radish peroxidase (1:2000, Abcam, Cambridge, MA, USA) were added for incubation at room temperature for 2 hours. Wash the membrane again. The images were observed after ECL solution (Amersham Bioscience, Uppsala, Sweden) was added. Scion Image analysis software (Scion Corporation, Frederick, MD, USA) was used to analyze the protein brand. The relative protein expression shall be the ratio of OD of target protein and β‐actin.

The total protein was extracted from the cells in each group or transplanted tumour tissue in nude mice via protein lysis buffer and quantified via the Bradford method (Thermo Fisher Scientific, Waltham, MA, USA). Protein (50 μg) was isolated for conducting sodium dodecyl sulphate polyacrylamide gel electrophoresis, which was then transferred into a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked via 5% skim milk powder for 1 hour, and mouse anti‐human REGγ (1:1000), Bcl2 (ab32124, 1:1000), Bax (ab32503, 1:1000), Caspase3 (ab32503, 1:1000), Cleaved‐Caspase‐3 (ab32042, 1:100) and β‐actin (1:1000, Abcam) were added for incubation at 4°C overnight. All antibodies were purchased from Abcam, Cambridge, MA, USA. The membrane was washed with PBST three times for 5 min, and rabbit anti‐mouse horseradish peroxidase‐labelled second antibody (1:2000; Abcam, Cambridge, MA, USA) was added for incubation at room temperature for 2 hours. Enhanced chemiluminescence (ECL; Amersham Bioscience, Uppsala, Sweden) was then added to the membrane, and the protein bands were analysed via the Scion image analysis system (Scion Corporation, Frederick, MD, USA). The relative amount of protein was expressed as the ratio of the OD value of the target protein to the β‐actin band.

Perihematomal brain tissues in ICH groups and corresponding area in sham group were collected for western blot anslysis. We extracted proteins of mitochondria, according to the manufacturer’s protocol from Isolation Kit for Tissue protocol (Pierce Biotechnology, Rockford, IL, United States). The detailed procedures of western was reported in one of our previous study.31 The primary antibodies were listed as follows: DJ-1 (1:15000, Abcam ab76008), p-Akt (1:2000, CST 4060s), Akt (1:3000, CST 4691), p-IKK (1:1000, CST 2697), IKK (1:1000, CST 2682), NF-κB p65 (1:3000, ab86299), Bax (1:1000, Abcam ab32503), Bcl-2 (1:500, Abcam ab59348), caspase-3 (1:500, Abcam ab13847), cleaved caspase-9 (1:1500, Abcam ab25758) and β-actin (1:5000, Abcam ab8226), NDUFS8 (1:2000, Abcam ab180183), COX IV (1:1000, CST 4844).

Proteins were extracted with RIPA buffer, uploaded on 10% SDS-PAGE gel, and transferred to PVDF membrane. Membrane was blocked with 5% non-fat milk for 2 h, and then incubated with antibodies (Abcam, Cambridge, CA, USA), including anti-SLC1A5 (1:1,000, ab237704), anti-Bcl-2 (1:1,000, ab32124), anti-Bax (1:1,000, ab32503), anti-YY1 (1:1,000, ab109228), anti-Nanog (1:200, ab21624), anti-Sox2 (1:1,000, ab97959), anti-GAPDH (1:2,500, ab9485), and secondary antibody (1:50,000, ab205718). ECL reagent (Beyotime) was then used for visualizing protein bands, and gray value was analyzed by Image J software.

Thymoquinone (TQ) was product of MCE (CAS: 490-91-5). Hoechst 33342 (B2261) was bought from Sigma-Aldrich. Cell counting kit‐8 (CCK‐8) was product of Selleck Chemicals (US). N-Acetyl-L-cysteine (NAC) and Z‐VAD‐FMK (ZVF) were obtained from Beyotime Biotechnology (Shanghai, China). The Apoptosis Detection Kit #556547 was purchased from BD (San Jose, CA). Antibodies against Bax (ab32503), Bcl‐2 (ab182858), LC3B (ab192890), Beclin-1 (ab207612), ATG7 (ab133528), MMP2 (ab92536), MMP9 (ab76003) and PD-L1 (ab213524) were bought from Abcam (San Francisco, CA). Antibody against vimentin (V6389) was purchased from Sigma-Aldrich (US). Antibodies against β‐actin (20536‐1‐AP) and Bcl-xl (10783-1-AP) were obtained from Proteintech (Chicago, IL). Antibodies against cleaved caspase 3 (9664), cleaved poly (ADP‐ribose) polymerase (PARP) (5625), E-cadherin (3195), N-cadherin (13116), and SQSTM1/p62 (8025) were obtained from CST company (NJ, US).

Proteins in H9c2 cells were extracted by lysis buffer (P0013J, Beyotime). After determining the concentration with a BCA detection kit (P0012, Beyotime), the proteins were loaded and electrophoresed on 10% SDS polyacrylamide gel (P0012AC, Beyotime), and then transferred onto PVDF membranes (FFP26, Beyotime,). After using 5% non-fat milk to block membranes for 60 min at 37°C, the following primary antibodies were incubated with membranes overnight at 4°C: Bcl-2 (ab59348, 1:1000, 26 kDa, Abcam, USA), Bax (ab32503, 1:1000, 21 kDa, Abcam), Cleaved caspase-3 (ab49822, 1:500, 17 kDa, Abcam), iNOS (ab3523, 1:200, 135 kDa, Abcam), Cox-2 (ab15191, 1:250, 69 kDa, Abcam), CDK4 (ab199728, 1:2000, 34 kDa, Abcam), Cyclin D1 (ab16663, 1:25, 36 kDa, Abcam), HSP70 (ab181606, 1:1000, 70 kDa, Abcam), and GAPDH (ab181602, 1:10000, 36 kDa, Abcam). Following extensive washing, protein bands were incubated with the secondary antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab205718, 1:2000, 42 kDa, Abcam) at 37°C for 2 h. The detection of signal was performed according to a standard ECL method (27), and analysis software (Image J 1.5i, National Institutes of Health, USA) was used for images to measured protein expression. GAPDH was used as housekeeping gene.

The protein level of TRIM16 was detected using Western blot, according to the experimental procedures recorded in the previous publications [29 (link)]. Briefly, transfected cells were lysed in Radioimmunoprecipitation assay (RIPA) lysis buffer with protease inhibitor (Thermo Fisher Scientific) to extract total protein. The protein quantification was measured by the BCA^TM Protein Assay Kit (Pierce, Appleton, WI, USA). Then the equivalent protein (50 µg) was added to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain the target protein. Next, the target proteins in the gel were transferred onto polyvinylidene difluoride (PVDF) membranes and incubated with primary antibodies against TRIM16 (ab72129, 1:2000, Abcam, Cambridge, MA, USA), hexokinase 2 (HK2, ab227198, 1:5000, Abcam), B-cell lymphoma protein 2 (Bcl-2)-associated X (BAX, ab32503, 1:2000, Abcam), Matrix metalloproteinase 2 (MMP2, ab92536, 1:1000, Abcam), Proliferating cell nuclear antigen (PCNA, ab18197, 1:1000, Abcam), and β-actin at 4°C overnight. The membranes were then incubated with second antibodies (Horseradish peroxidase-conjugated IgG antibody). Finally, the Western blot intensities were detected using LAS 4000 Image Reader (Fujifilm, Tokyo, Japan)