β actin

The extraction of total hairy root protein was carried out using a Plant Protein Extraction Kit (CoWin Biosciences, Beijing, China). The extracted proteins were assayed by a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Massachusetts MA, USA). Standard Western blots were performed. Immunoblotting was carried out by using the following primary antibodies: GSH-S (Agrisera AB, Vännäs, Swedish), GST class-phi (Agrisera AB, Vännäs, Swedish), and β-actin (CoWin Biosciences, Beijing, China). After incubation with the secondary antibodies, the signal was developed by chemiluminescence and autoradiography. Densitometric analysis was performed using ImageJ software (National Institutes of Health, Bethesda, NY, USA).

Western blot analysis was performed as described in our previous studies [7 (link), 9 (link), 10 (link)]. RIPA lysis buffer extracted proteins, and then, G250 protein assay (Beyotime, Jiangsu, China) extracted soluble protein. Protein samples were loaded on 10% sodium dodecyl sulfate polyacrylamide gels (Invitrogen), transferred to polyvinylidene fluoride membranes (Bio-Rad), and blocked with 5% nonfat milk powder. Membranes were incubated overnight with the following primary antibodies for human Runx2 (Cell Signaling, Beverly, MA, USA), OCN (Santa Cruz, Dallas, TX, USA), NF-κB (Santa Cruz), p-NF-κB (Santa Cruz), and β-actin (Cowin Biotech, Beijing, China). Torseradish peroxidase- (HRP-) conjugated secondary antibody (Cowin Biotech) incubated the membranes for 2 h at room temperature. Protein signals were visualized by using the ECL Western Blotting Detection System (GE Healthcare, Piscataway, NJ, USA).

Cells were lyzed with a lysis buffer containing phenylmethyl sulfonylfluoride (PMSF) (Beyotime, Shanghai, China) at 4°C. Proteins were quantified using a BCA protein assay kit (ComWin Biotech, Beijing, China). Protein lysates (50 μg) were separated by 10% SDS-PAGE gels (Invitrogen) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Beyotime, Shanghai, China). The membranes were blocked with 5% non-fat dry milk in Tris-phosphate buffer containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature and then treated with the primary antibodies at 4°C overnight. After washing with TBST, the membranes were incubated with peroxidase-conjugated affinipure goat anti-rabbit IgG (H+L) (ZSGB-BIO, 1:5000, ZB-2301) and peroxidase-conjugated affinipure goat anti-mouse IgG (H + L) (ZSGB-BIO, 1:5000, ZB-2305) for 1 h at room temperature. The blots were visualized using an ECL kit (ComWin Biotech, Beijing, China), and quantified using the Image J Software, normalized to β-actin. The primary antibodies were: E-cadherin (1:500, Cell signaling, #3195), Vimentin (1:1000, Cell signaling, #5741), ERK1/2 (1:1000, Cell signaling; #4695), p-ERK1/2 (Thr202/Tyr204) (1:1000, Cell signaling, #9101), α-catenin (1:500, Proteintech, Catalog number: 66221-1-Ig), β-catenin (1:1000, Santa Cruz, sc-7963), and β-actin (1:1000, Bioss).