Protease and phosphatase inhibitor cocktail

SDS-PAGE and western blot investigations were performed by previously developed protocol with slight modifications [19 (link), 20 (link)]. Briefly, after 24 h, and 48 h of treatments with NVD at vital doses, HCT116 and HT29 cells were lysed in DMEM augmented with freshly added protease and phosphatase inhibitor cocktail 1:100 (Santa Cruz, CA) and protein concentration were estimated by Bradford assay [21 ]. For western blotting 8–12% polyacrylamide gels were used to resolve 40 μM of protein, transferred on to a nitrocellulose membrane, probed with appropriate monoclonal primary antibodies, and detected by super signal west Pico, Dura or Femto Chemiluminescence Reagent (Thermo Scientific, USA). Quantification of protein bands was done through measuring band density using Image J software. The densities of the bands (normalized to actin) relative to that of the untreated control (designated as 1.00) were presented as mean ± SEM of three separate experiments.

Cells were lysed on ice using RIPA lysis buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS, 10 mM Tris and 150 mM NaCl) with protease and phosphatase inhibitor cocktail (Santa Cruz Biotechnology Inc, Heidelberg, Germany). After lysis, cell lysates were split by ultrasonic (Sonics, China, Cat#VCX130) and centrifuged at 14,000 g for 5 min at 4 °C. The protein concentration of supernatant was measured using a BCA protein assay kit (Beyotime, China). The reduced proteins (30 μg) in 4 × sample buffer (Invitrogen) and β-2-mercaptoethanol were heated at 95 °C for 5 min before loading. Protein samples were separated on 10% gel and transferred to nitrocellulose filter membranes. The membranes were blocked with 5% non-fat dry milk (Bio-Rad, Calif, USA) in TBS-T, and incubated with primary antibody overnight at 4 °C, followed by the fluorescent secondary antibodies for 1 h at room temperature. After washing, membranes were scanned using the Odyssey infrared imaging system (LI-COR Biosciences, USA). Densitometric analysis was done using Image J software (NIH) to quantify protein expression levels with GAPDH or tubulin as internal control.

Mouse hearts were homogenized and lysed on ice in RIPA lysis buffer supplemented with protease and phosphatase inhibitors (1 mM Na₃VO₄, 10 mM NaF, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 5 μg/ml aprotinin, 5 μg/ml leupeptin). Cultured cell samples were homogenized in RIPA lysis buffer containing protease and phosphatase inhibitor cocktail (Santa Cruz). The protein concentration was determined using the bicinchoninic acid (BCA) reagent according to the manufacturer’s instructions (Thermo Fisher Scientific). 30 μg of protein was separated by 10-15% SDS-PAGE gel and then transferred to nitrocellulose membranes (Bio-Rad). After blocking with 5% BSA in Tris-buffered saline/Tween-20 (TBST) for 1 h at room temperature, blots were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: MMP-9 (1:500, Abcam), p-p38 MAPK (1:1000, Cell Signaling), p38 MAPK (1:1000, Cell signaling), p-JNK (1:1000, Cell Signaling), JNK (1:1000, Santa Cruz), p-ERK (1:1000, Cell Signaling), ERK (1:1000, Cell Signaling), HSP90 (1:3000, BD Biosciences). Immunoblots were then incubated with corresponding secondary antibodies (1:5000, LI-COR) for 1 h at room temperature and developed using ODYSSEY Infrared Imaging System (LI-COR). HSP90 was used as a loading control. Data were quantified with Image Studio software (LI-COR, Version 5.2).