Anti β actin sc 8432

The human colorectal cancer cell lines HCT116 and SW480, and mouse colon carcinoma cell line CT26 were cultured in a complete RPMI 1640 growth medium (Thermo Fisher Scientific, CA, USA) with 10% fetal bovine serum (Invitrogen, CA, USA), 3.5 g/L glucose (Thermo Fisher Scientific, CA, USA), 10 mM HEPES (Thermo Fisher Scientific, CA, USA), and 1.0 mM sodium pyruvate (Thermo Fisher Scientific, CA, USA) at 37 °C in an incubator of 5% CO₂ and 95% air.
The following antibodies were used in this study: anti-PD-L1 (ab205921, clone 28-8, Abcam, Cambridge, UK) anti-PD-L1 (#13684, clone E1L3N, Cell Signaling Technology, MA, USA), anti-β-actin (sc-8432, Santa Cruz, CA, USA), anti-DNMT3a (sc-365769, Santa Cruz, CA, USA), anti-DNMT1 (sc-271729, Santa Cruz, CA, USA), anti-p-signal transducer and activator of transcription 1 (p-STAT1, sc-8394, Santa Cruz, CA, USA), anti-STAT1 (sc-464, Santa Cruz, CA, USA), anti-interferon regulatory factor 1 (IRF1, sc-514544, Santa Cruz, CA, USA), and horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz, CA, USA).
Lentiviruses carrying individual shRNA were obtained from the National Core Facility for Manipulation of Gene Function by RNAi, miRNA, miRNA sponges, and CRISPR/Genomic Research Center, Academia Sinica, Taipei, Taiwan.

Protein extracts were prepared with RIPA buffer containing a protease inhibitor mixture and protein concentrations were determined using BCA Protein Assay. Proteins were transferred to the membrane and blocked with 5% skim milk for 1 h after being separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific primary antibody: anti-TFAM (ab176558, 1:1000, Abcam, Cambridge, MA, USA), anti-PPARβ (ab178860, 1:1000, Abcam, Cambridge, MA, USA), anti-KLF16 (sc-377,519, 1:500, Santa Cruz Biotechnology) and anti-β-Actin (sc-8432, 1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to incubate with the membrane overnight at 4°C. After being washed with tris buffered saline-tween (TBST) for 15 min, the secondary antibody (sc-2005, sc-2006, 1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to incubate the membrane for 2 h at room temperature. Protein bands were visualized via enhanced chemiluminescence (Thermo Fisher, USA) and quantified using ImageJ software (version 1.48 v, NIH, Bethesda, MD).

Cells were lysed with IP lysis buffer containing a 10% cocktail (B14001, Bimake, Houston, TX, USA), and the protein concentration was determined using a BCA kit (Pierce Chemical, Rockford, IL, USA). SDS-PAGE was performed using 30–50 μg of total cell protein. Then, the protein was transferred to a PVDF membrane, blocked with 5% skim milk at room temperature, and incubated with the indicated antibodies. The primary antibodies used were anti-LMP1 (M0897, 1:500) purchased from DAKO (Glostrup, Denmark), and anti-LC3 obtained from NOVUS (NB100-2220, 1:500, Littleton, CO, USA). Anti-BNIP3 (ab10433, 1:1000) and anti-Beclin1 (ab207612, 1:1000) were purchased from Abcam (Cambridge, MA, USA). Anti-p-Akt (ser473, 9270, 1:1000), anti-p-ERK1/2 (4370, 1:1000), anti-p-JNK (Thr183/Tyr185, 9251, 1:1000), anti-p-JAK3 (Tyr980/981, 5031, 1:1000), anti-rabbit IgG-HRP (14708, 1:2000), and anti-mouse IgG-HRP (14709, 1:2000) antibodies were obtained from cell signaling technology (Danvers, MA, USA). Anti-Bcl-2 (sc-7382, 1:500), anti-p-P38 (thr180/try182, sc-4511, 1:1000), anti-HIF1α (sc-53546, 1:500), and anti-β-actin (sc-8432, 1:2000) antibodies were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA). Blots were analyzed using a chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA).