Bca protein assay kit

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).

The total proteins of cells or mitochondrial proteins were collected using RIPA lysis buffer (Beyotime, Beijing, China) on ice. The cytoplasmic and mitochondrial proteins were gathered using a Mitochondria Isolation Kit (Kaiji Biotechnology Co., Ltd., Jiangsu, China). Protein concentration was determined by BCA Protein Assay Kit (ComWin Biotech Co., Ltd., Beijing, China). Protein samples (12–15 μg) were electrophoresed on 10–12% gradient SDS-PAGE gel. Then, electrophoretically separated proteins were transferred to PVDF membranes. The membranes were closed and incubated with antibodies against LC3II, p62, ATG5, Beclin-1, Parkin, GAPDH, and TOMM20 at 4 °C overnight, followed by goat anti-mouse IgG and goat anti-rabbit IgG antibodies. The membrane was visualized using chemiluminescent fluid and ImageJ software for quantitative analysis.

MSCs were cultured with the conditioned media mentioned above for 48 h and EPCs were co-cultured with the conditioned media for 4h, as long as the tube formation assay. Then the cells were lysed in buffer and total protein contents were determined using the BCA protein Assay Kit (ComWin Biotech Co. Ltd, China). Equal amounts of protein were subjected to SDS-PAGE and transferred to PVDF membranes (Merk Millipore, USA). The membranes were blocked with 5% non-fat milk, probed with anti-VEGF (ABS82, polyclonal, Merk Millipore, USA), anti-PDGFRβ (MA5-15143, monoclonal, Thermo Fisher Scientific, USA) for EPCs and anti-RUNX2 (ab23981, polyclonal, Abcam, USA), anti-OCN (ab93876, polyclonal, Abcam), anti-PDGFRβ for MSCs. The results were normalized to the expression level of β-actin (ab3280, monoclonal, Abcam). Then the membranes were stained with horseradish peroxidase-coupled secondary antibodies. Protein bands were visualized using enhanced chemiluminescence (GE Healthcare Life Sciences, UK).

Total protein was extracted with RIPA buffer and the protein concentration was quantified using a BCA protein assay kit (Com Win Biotech, Qingdao, China). A total of 50 μg protein was separated by 10% SDS-polyacrylamide gel electrophoresis, and then transferred onto the Clear Blot membrane-p (ATTO, Tokyo, Japan). The blots were incubated with anti-E-Cadherin (ab1416), anti-Vimentin (ab8978), anti-GAPDH (ab181602) (all 1:1000; Abcam, Shanghai, China), and anti-NRBP1 (Cat# H00029959-M01, 1:1000, Abnova, Taipei, Taiwan) overnight at 4 °C and then incubated with HRP-labeled specific secondary antibody for 1 h at room temperature. Protein blots were visualized with an enhanced chemoluminescence kit (KeyGen) and scanned using ImageJ software based on the mixture of both intensity and band thickness.

The cells were suspended in the appropriate lysis buffer. Following centrifugation (10,000 rpm for 10 min), the protein content in the supernatants was quantified using the BCA protein assay kit (Beijing ComWin Biotech Co., Ltd., Beijing, China). The boiling denatured proteins were segregated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore, Billerica, MA, USA) at 4°C. The membranes carrying proteins were incubated with primary antibody and detected with the appropriate secondary polyclonal antibody by BeyoECL Plus (Beyotime Institute of Biotechnology, Jiangsu, China). Antibodies against PKR and NLRP3 were used to precipitate proteins from cell lysis in the presence of 20 μl protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. Protein complexes were washed 4 times with lysis buffer, and then incubated at 95°C for 5 min and resolved by western blot analysis.

Total proteins were extracted from apple calli, apple fruit and tomato fruit using the following extraction buffer (pH 7.5): 100 mM Tris–HCl, 100 mM KCl, 10% glycerol, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1% Triton X‐100 and 1× protease inhibitor cocktail. Protein concentration was measured using a BCA Protein Assay Kit (ComWin Biotech, Beijing, China) and adjusted to the same level for each sample.