Bca protein estimation kit

Post-garcinol treatment, C6 cells were pelleted and lysed in chilled RIPA buffer and were supplemented with a phosphatase and protease inhibitor (Sigma-Aldrich, Missouri, United States) cocktail for 30 min. A BCA protein estimation kit (Sigma-Aldrich, Missouri, United States) was used to quantify the protein concentration in various groups. The proteins were then separated through SDS-PAGE and subsequently transferred onto PVDF membranes (Millipore Corporation, Massachusetts, United States). Non-specific binding was prevented by incubating the PVDF membrane with blocking buffer, constituted by 5% non-fat milk for 2 h. Thereafter, the membrane was incubated overnight with primary antibodies at 4 °C. Post-incubation, the membrane was again incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature and were then washed with TBST buffer. The expression of NF-κB, cleaved caspase-9, cleaved caspase-3, and surviving genes were evaluated by detecting changes in the chemiluminescence levels using an ECL kit (Sigma-Aldrich, Missouri, United States) following the manufacturer’s manual. Subsequently, the level of protein expression was normalized to relative internal standard and further quantified using ImageJ software (National Institutes of Health, Maryland, United States).

For western blot analysis, lysates were prepared by harvesting cells in lysis buffer [20 mM Tris pH 7.2, 5 mM EGTA, 5 mM EDTA, 0.4% (w/v) SDS and 1X protease inhibitor cocktail]. Protein was quantified with a BCA protein estimation kit (Sigma). Total protein samples (∼40 µg) were analyzed on 12% polyacrylamide gels, followed by immunoblot analysis using a standard protocol. In brief, proteins were transferred to nylon membrane, which was blocked with TBS-T buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20) containing 5% skim milk powder. The blots were washed with TBS-T buffer and incubated (overnight, 4°C) in the same buffer with primary anti-PPARγ, -PTEN, -survivin, -Bcl-2, -Bax caspase-9 (1∶500) or -β-actin (1∶1000) antibodies (all from Santa Cruz Biotechnology). Blots were then washed and incubated with HRP (horseradish peroxidase)-conjugated anti-rabbit or -mouse secondary antibody (1∶20,000). Color was developed in the dark using the ECL kit (GE Healthcare, Bucks, UK) and blots were analyzed by densitometry with ImageJ 1.43 using β-actin as internal control.

Protein was extracted from CD44v6 knockdown and 5FU+ Silibinin treated CD44+ HCT116 cells and the concentration was determined by BCA protein estimation kit (Sigma-Aldrich, USA). 50 μg of protein was resolved on 12% SDS-PAGE and transferred to PVDF membrane by wet electro-blotting method at 4 °C. The membrane was blocked with 5% non-fat milk in Tris buffered saline for 3 h at room temperature followed by overnight incubation with primary the primary antibodies: anti-CD44v6 (1:250), anti-PARP (1:1000), LC-3 (1:1000) and anti-β-actin (1:1000) at 4 °C. Membrane was probed with horseradish peroxidase (HRP) conjugated secondary antibodies (1:10,000). Proteins were detected using EZ-ECL kit (Clarity Western ECL substrate, BIO-RAD, USA) according to manufacturer’s instructions and signal was captured on Kodak X-Omat blue film (NEN Life Sciences, Inc., Boston, MA) in the dark^{54 (link)}.

In brief, 2 × 10⁶ cells were subjected to treatments as mentioned in the figure legends. The cells were lysed in NP-40lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 0.2% Nonidet P40, and 1X protease inhibitor mixture (Roche, Basel, Switzerland) and the protein concentration was estimated by the BCA protein estimation kit (Sigma, St. Louis, MO, USA). Next, 350 µg of protein were incubated with 2 µg of respective antibodies (as mentioned in the figure legends) with 20 µL of SepharoseA/G beads overnight at 4 °C with gentle shaking. The beads were washed with RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, 1% triton-X 100, 0.5% Na-deoxycholate, 0.1% SDS, and 1X protease inhibitor mixture) and the protein samples were fractionated on 8–12% SDS-PAGE followed by Western blot.

Molecular biology grade reagents were commercially purchased. Thiazolyl Blue Tetrazolium Blue (MTT), trypan blue dye, H₂DCFDA, 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI), DEAE-Dextran, 7,12-dimethyIbenz[a]anthracene (DMBA), and BCA protein estimation kit were purchased from Sigma–Aldrich (St. Louis, MO, United States). Dulbecco’s Modified Eagle’s Medium (DMEM), Leibovitz L-15 medium (L-15), Dulbecco’s Phosphate buffer saline (DPBS), and fetal bovine serum (FBS) were purchased from Invitrogen (Life Technologies, United States). Estrogen (ab32063), Progesterone (ab2765), HER2 (ab106575), CD31 (ab28364), ki67 (ab15580), p53 (ab131442), p63 (ab124762), CK5/6 (MA5-12429), bcl2 (ab59348), PCNA (ab18197), b-catenin (ab32572), and E-cadherin (ab133597) antibodies were purchased from Abcam, United Kingdom. β-Interferon (Relibeta) was purchased from Reliance, Pvt. Ltd., β-interferon ELISA kit was purchased from YH Biosearch Laboratory (China), Annexin V and propidium iodide were purchased from Thermo Fisher Scientific (United States). All other chemicals used were of analytical grade and purchased from Merck (Darmstadt, Germany).

To analyze the DISC and binding association of FADD or cFLIP_L to RIP1 during FADD expressed cells or cFLIP_L knockdown condition co-immunoprecipitation was carried out. In brief, 2 × 10⁶ HEK 293T cells were seeded in 60 mm dishes followed by treatments as described in the figure legends. The cells were lysed in 1 ml of lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 0.2% Nonidet P40, and 1X protease inhibitor mixture (Roche, Switzerland) for 30 min on ice. The total cell lysate was centrifuge at 15,000 g for 15 min at 4 °C. The supernatant was collected and protein concentration was determined by the BCA protein estimation kit (Sigma, USA). Next, 300 μg of protein was incubated with 20 μl of Protein A-Sepharose beads (BioVision, USA) for 1 h at 4 °C with gentle shaking. The mixture was spun down at 3000 rpm for 5 min at 4 °C and the collected supernatant was incubated with 1 μg of respective antibodies and 20 μl of Protein A-Sepharose beads for overnight at 4 °C with gentle shaking. The beads were washed three times with 1 ml of RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, 1% triton-X 100, 0.5% Na-deoxycholate, 0.1% SDS and 1X protease inhibitor mixture) and finally resuspended in 6X Laemmli sample buffer. The protein samples were fractionated on 12% SDS-PAGE followed by western blot as described above.

Molecular biology-grade reagents were purchased commercially. Poly-L lysine, protease inhibitor cocktail, H₂DCFDA, DAPI, Flouramaount, SMCC (Succinimidyl- trans-4-(N-maleimidylmethyl)cyclohexane-1-carboxylate), HIV-TAT1, Iodocetamide, DMF, Cyclodextrin, Cycloheximide (CHX), Etoposide, Methyl β-cyclodextrin (MβCD), anti-His (SAB4301134), trypan blue, and a BCA protein estimation kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ni⁺²-NTA beads, LipofectamineLTX plus transfection reagent Prestoblue viability assay kit, IL-1 beta Human ELISA Kit (BMS224HS), and Alexa fluors were purchased from Invitrogen (Life Technologies, Carlsbad, California, USA). A dual glow luciferase assay kit was purchased from Promega (Madison, WI, USA). The death receptor ligands CD 95L and TNF-α were obtained from ProSpec (Rehovot, Israel). HA14-1 was obtained from Maybridge (Cornwall, UK). The details of the antibodies used in this study are provided in Supplementary Material Table S1. All other chemicals used were of analytical grade and purchased from Merck (Darmstadt, Germany).