Antibody against actin

High-glucose DMEM media were purchased from Gibcol (Grand Island, NY, USA). Cardamonin was isolated from the dried seeds of Alpinia katsumadai Hayata. (Anhui, China). The purity of cardamonin is > 99% in HPLC analysis. Rapamycin, bovine serum albumin and MTT were from Sigma (St Louis, MO, USA). The 10 mM solution of cardamonin and 100 μM solution of Rapamycin were prepared in DMSO, stored at -20°C, and then diluted as needed in cell culture medium. Rabbit antibodies against mTOR, p-mTOR (Ser2448), S6K1, p-S6K1 (Thr389), E-cadherin, Snail and the secondary antibody (anti-rabbit IgG, HRP-linked antibody) were from Cell Signaling Technology (Boston, MA, USA). Antibody against actin was from Santa Cruz Biotechnology (Dallas, TX, USA). Matrigel was purchased from BD Biosciences (San Jose, CA, USA).

Cell lysates were prepared in RIPA buffer (Sigma) with protease inhibitor cocktail (Roche). 30 μg of total protein was resolved on a 12% SDS polyacrylamide gel and probed with anti-Bim, anti-Noxa and anti-Mcl-1. Antibody against actin (Santa Cruz Biotechnology, Santa Cruz, CA) was used as a loading control.

Total proteins were extracted from HM of colon and colon tumor tissue (TT) of affected patients and from primary colon cancer cultures, using QIAzol Reagent (Qiagen) following the manufacturer’s instructions. Concentrations were determined by using a protein assay kit adopting bovine serum albumin standards, according to the manufacturer’s instructions (Bio-Rad Laboratories, Inc.). A total of 30 μg of proteins were separated by SDS-polyacrylamide gel electrophoresis and blots were prepared on a Amersham Hybond-ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Inc./GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). The primary antibody against β-catenin, E-cadherin (monoclonal, rabbit anti-human; no. 3195), CD44 (monoclonal, mouse anti-human; no. 5640), Snail (monoclonal, rabbit anti-human; no. 3879) and vimentin (monoclonal, rabbit anti-human; no. 5741) was from Cell Signaling Technology, Inc. (Beverly, MA, USA). The antibody against actin (polyclonal, rabbit anti-human; no. sc-1615) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The membrane was probed with a secondary antibody against peroxidase-conjugated rabbit or goat immunoglobulin G, and immunoreactive bands were detected using the enhanced chemiluminescence Immobilon Western HRP Substrate (Millipore, Billerica, MA, USA).

Western blot analysis in whole cells and the mitochondrial fraction was performed as previously described [4 ,22 (link)]. Intracellular renin (int-renin) was assessed with primary antibody against renin, which was purchased from AnaSpec (Fremont, CA, USA). Primary antibodies against Phosphorylated-ERK1/2, ERK1/2, phosphorylated-Akt (Ser473), Akt, phosphorylated-p38, and p38 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibody against actin was acquired from Santa Cruz Biotechnology (Dallas, TX, USA), prohibitin antibody was from Abcam (Cambridge, UK), and horseradish peroxidase-conjugated secondary antibodies were from Promega (Madison, WI, USA). Immunoblotting was performed using an enhanced Can Get Signal kit (TOYOBO, Osaka, Japan). Densitometric analysis was performed using the Molecular Imager ChemiDoc™ system (Bio-Rad Laboratories, Hercules, CA, USA).

Cisplatin, doxorubicin and VX‐765 were purchased from Selleck. Ac‐DEVD‐CHO was obtained from MedChem Express. Propidium iodide (PI), Hoechst 33342, dimethyl sulfoxide (DMSO) and Tween‐20 were bought from Sigma‐Aldrich. Lipofectamine RNAiMAX, Dulbecco's Modified Eagle's Medium (DMEM) medium with high glucose, Opti‐MEM, foetal bovine serum (FBS), streptomycin and penicillin were the products of ThermoFisher. The antibody against actin was purchased from Santa Cruz. The antibodies against cleaved caspase‐3, caspase‐3, cleaved caspase‐7, cleaved caspase‐8, cleaved caspase‐9, PARP and horse‐radish peroxidase (HRP)‐linked IgG were purchased from Cell Signaling Technology. Antibodies against pro‐caspase1+p10+p12, GSDMD (clone: EPR19828) and DFNA5/GSDME were obtained from Abcam. F4/80‐PE, Ly‐6C‐APC and CD11b‐AlexaFluor488 were obtained from eBioscience. F4/80‐AlexaFluor647 was bought from BioLegend.

6-OHDA was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). CellTiter-Glo^® 2.0 was purchased from Promega Corporation (Madison, WI, USA). Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DMEM/Ham’s-F12) and low-glucose DMEM were purchased from Fujifilm Wako Pure Chemical Corporation (Tokyo, Japan). FastGene™ RNA Basic kit was obtained from Nippon Genetics Co., Ltd. (Tokyo, Japan), PrimeScript^™ RT master mix (Perfect Real Time) was obtained from Takara Bio (Shiga, Japan), and THUNDERBIRD^® Next SYBR^® qPCR mix was obtained from Toyobo (Osaka, Japan). 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) was obtained from Merck KGaA (Darmstadt, Germany), and MitoSOX™ was obtained from Thermo Fisher Scientific (Waltham, MA). Antibody against actin (Code: SC-47778) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phospho-JNK (p46 and p54) (Code: #4668), phospho-p38 (Code: #4511), phospho-ERK (Code: #4376), and IκBα (Code: #4814) and goat anti-rabbit IgG, HRP-linked antibody (Code: #7074) were purchased from Cell Signaling Technology Japan (Tokyo, Japan). HRP-conjugated donkey anti-mouse IgG was purchased from GE Healthcare (Tokyo, Japan).