Bca protein assay kit

Protein was extracted from HT29 cells using ice-cold RIPA buffer (Elabscience Biotechnology, Inc.) and determined by using a BCA protein assay kit (Phygene). Protein samples were subjected to separation via 10% SDS-PAGE (DetaiBio Tech) and transferred onto PVDF membranes (Roche Diagnostics), after which time the membranes were blocked with 5% non-fat milk for 2 h at room temperature. The membranes were incubated at 4˚C overnight with the following primary antibodies (all Abcam): Nrf2 (1:1,000, cat. no. ab137550), heme oxygenase-1 (HO-1; 1:2,000, cat. no. ab52947), NADPH dehydrogenase quinone 1 (NQO1; 1:10,000, cat. no. ab80588), GPX4 (1:1,000, cat. no. ab125066), ferritin heavy chain 1 (FTH1; 1:1,000, cat. no. ab183781), transferrin (1:1,000, cat. no. ab277635) and GAPDH (1:1,000, cat. no. ab8245). Following primary antibody incubation, the membranes were incubated with HRP-conjugated secondary antibodies (goat anti-mouse IgG H&L, 1:2,000, cat. no. ab6789; or goat anti-rabbit IgG H&L, 1:2,000, cat. no. ab6721) for 2 h at room temperature. An ECL Western Blotting Substrate kit (AmyJet Scientific, Inc.) was applied to visualize protein bands. The resultant images were analyzed using ImageJ software (version 1.8.0; National Institutes of Health) and the quantification of each group was performed in triplicate.

Total proteins were extracted using a protein extraction kit (Phygene). A BCA Protein Assay Kit (Phygene) was used to quantify the concentration of protein. After proteins were denatured at 95°C for 10 min, a Tricine‐SDS‐PAGE Gel Kit (Phygene) was used to prepare the SDS‐PAGE gels separating protein bands. The proteins were transferred onto nitrocellulose membranes (Roche) and immersed in 5% defatted dry milk to block aspecific signals. The membranes were incubated with anti‐matrix metalloprotein 2 (anti‐MMP2, 1:2000; Cusabio Biotech), anti‐MMP9 (1:800; Cusabio Biotech), anti‐RAB3D (1:1000; Affinity), and anti‐β‐actin (1:5000; Affinity). Subsequently, the membranes were washed using Tris buffered saline Tween (Millipore) and incubated with horseradish peroxidase‐labeled secondary antibody (1:5000; Affinity). Biuret Reagent (A + B) (Phygene) was used to develop protein blots. β‐actin served as a control.