Hrp labeled secondary antibody

Anti-Flag beads (Cat. # M8823, Sigma Aldrich) were used to immunoprecipitate target protein from the cell culture medium. Western blotting was performed as described previously [17 (link)]. In brief, total proteins were extracted by RIPA. After SDS-PAGE, samples were transferred to the PVDF membrane (Millipore, USA) with transfer membrane apparatus (BIO-RAD, USA). The membrane was blocked by 5% milk in blocking buffer for 2 h. Wash the membrane with a washing buffer 3 times. Anti-Flag (Cat.# F1804, Sigma Aldrich) and anti-β-Actin (Cat.# ab8227, Abcam) primary antibody was added and incubated at 4 °C overnight. The HRP-labeled secondary antibody was added (1:2000, Abcam) to incubate at room temperature for 2 h and then washed 3 times. Luminata TM Crescendo Western HRP Substrate was added. Immunoreacted bands were captured by an enhanced chemiluminescence system (BIO-RAD, USA).

RIPA lysis buffer (R0010, Solarbio, China) was adopted to harvest total protein of tissues or cells. Afterward, protein concentration was measured with a BCA kit (Yeasen, Shanghai, China) before electrophoresis separation based on sodium dodecyl sulfate–polyacrylamide gel. Then, the treated protein was loaded onto a polyvinylidene fluoride membrane which was then combined with the primary antibody at 4 ℃ overnight and with the diluted HRP-labeled secondary antibody (1:20,000, Abcam) for 1 h. Finally, enhanced chemiluminescence-developed protein signals were quantified using Image J analysis software. Primary antibodies: cleaved Caspase-3 (9664S, Cell Signaling Technology), Bcl‑2 (15071S, Cell Signaling Technology), HK2 (22029-1-AP, Proteintech), PKM2 (4053, Cell Signaling Technology), and GAPDH (ab8245, Abcam).

Total protein extractions were performed with lysis buffer (Thermo Fisher Scientific, Inc.). The BCA Protein Assay Kit (Thermo Scientific) was utilized to measure the protein concentrations. An equal amount of protein samples were subjected to 10% SDS-PAGE, followed by transfer onto PVDF membranes (Invitrogen). After being blocked with 5% skimmed milk, the membrane was then incubated at 4°C overnight with primary antibodies against E-cadherin (1 : 2000, Abcam), N-cadherin (1 : 2000, Abcam), vimentin (1 : 1000, Abcam), PI3K (1 : 1000, Abcam, Cambridge, MA, USA), p-PI3K (1 : 2000, Abcam), AKT (1 : 1000, Abcam), p-AKT (1 : 1000, Abcam), and GAPDH (1 : 1000, Abcam). After that, the membranes were probed with appropriate HRP-labeled secondary antibody (1 : 3,000, Abcam) for 2 h at room temperature. Finally, the protein band was visualized with ECL reagents (Millipore, USA). GAPDH was an internal control.

Total proteins were extracted using Radio-immunoprecipitation assay (RIPA) lysis buffer containing phenylmethanesulfonyl fluoride (PMSF; Solarbio, Beijing, China). Protein concentration was measured using bicinchoninic acid kit (Yeasen, Shanghai, China). After sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was transferred to the polyvinylidene fluoride membrane, blocked with 5% skimmed milk powder and added with primary antibodies DLC1 (1:100, Santa Cruz Biotechnology), EZH2 (1:1000) and GAPDH (1:2500, both from Abcam). Subsequently, the membrane was incubated with HRP-labeled secondary antibody (1:2000, Abcam) and visualized by the enhanced chemiluminescence kit (Ameshame, UK). The images were captured by the system (Bio-Rad, CA, USA) and data were analyzed by Quantum One v4.6.2 software.

Melanoma tissue and paracancerous tissue were collected and fixed in paraformaldehyde at room temperature, rinsed with PBS for 3 times, and embedded in paraffin and sectioned. The sections were dewaxed with xylene-ethanol solution, followed by sodium citrate buffer (pH 6.0) for antigen repair, and rinsed with PBS for 3 times. They were put in 30% hydrogen peroxide solution and reacted for 30 minutes in dark at room temperature. They were rinsed with PBS for 3 times. They were blocked with 3% BSA at room temperature for 20 min, then, Axl antibody (Abcam) or Akt antibody (Abcam) was added and incubated overnight in the refrigerator at 4°C. They were rinsed with PBS for 3 times, and HRP-labeled secondary antibody (Abcam) was added at the appropriate concentration and incubated at room temperature for 30 min. They were rinsed with PBS 3 times, 5 minutes each time. DAB chromo-developing solution (Solarbio®, Life Science) was added for staining for 2 min, and sections were rinsed with running water. The hematoxylin solution was redyed for 2 min and rinsed with PBS for 15 min. The slides were dehydrated in ethanol, then, 80% glycerin was added to the slides, and the cover glass was sealed. Finally, microscope observation was performed and photographs were taken (magnification: ×200).