Ecl kit

Proteins were collected using lysis buffer (Beyotime Biotechnology, China) containing protease inhibitor (PMSF, 1:100) and were centrifuged at 20627g for 20 min at 4°C; the supernatant was subsequently transferred to new tubes. Protein concentrations were detected using a BCA protein kit (Beyotime). lysis buffer was mixed with protein loading buffer and protein was denatured at 100°C for 5 min. An equal amount of proteins was separated using SDS-polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Membranes were blocked for 2 h with 5% skimmed milk and incubated with primary antibodies overnight at 4°C. Secondary antibodies were subsequently added for 1.5 h at room temperature. Protein bands were obtained using an enhanced chemiluminescence (ECL) kit (Servicebio, Wuhan, China) using GENESys (Synoptics Ltd, China). anti-SMAD7 (#25840-1-AP) was purchased from Proteintech (China). anti-YAP1 (#14074), anti-E-cad (#3195), anti-N-cad (#13116), anti-vimentin (#5741) and anti-CTGF (#86641); these antibodies were purchased from Cell Signaling Technologies (USA). Anti-Bcl2 (#ab182858), anti-Bax (#ab32503) and anti-GAPDH (#ab8245) antibodies were purchased from Abcam (UK) while Goat Anti-Rabbit IgG HCS (#A25222) and goat anti-mouse IgG-horseradish peroxidase (#A25012) were purchased from Abbkine (USA).

Proteins were extracted from cell or tissue samples using a protein extraction buffer and subsequently separated by SDS-PAGE. Following electrophoresis, the proteins were transferred onto a polyvinylidene difluoride membrane using the wet transfer method. The membrane was then blocked with 5% non-fat milk to prevent nonspecific binding and subsequently probed with a specific primary antibody targeting the protein of interest overnight at 4 °C. After thorough washing with TBST to remove the unbound primary antibody, the membranes were incubated with a secondary antibody for 1 h at room temperature. The ECL kit (Servicebio Technology) was used to visualize the membranes. The sources of the primary antibodies were as follows: GAPDH (Mouse, 60004Ig, 1:10,000, Proteintech, Wuhan, China), BMP2 (Mouse, A0231, 1:2000, Proteintech, Wuhan, China), OCN (Rabbit, A20800, 1:1000, ABclonal, Wuhan, China), RUNX2 (Rabbit, AF4770, 1:2000, Affinity, Jiangsu), ALP (Rabbit, AF4770, 1:2000, Affinity, Shanghai, China). The source of the secondary antibody is as follows: HRP-Goat anti Rabbit (Searcare, 5220-0336, 1:50,000), HRP-Goat anti Mouse (Searcare, 5220-0341, 1:50,000).

We performed Western blot analysis as previously described (3). Tibial growth plate specimens were centrifuged, and the supernatants were separated by SDS-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane (Millipore). The membranes were incubated with rabbit-source antibodies to FGF2 (Abclonal Technology, #A0235, 1:1,000), FGFR1 (Abclonal Technology, #A2073, 1:1,000), VEGFA (Abcam, #ab46154, 1:1,000) and VEGFR1 (Abcam, #ab32152, 1:1,000), and then re-probed with appropriate horseradish peroxidase-conjugated secondary antibodies (Servicebio technology, #GB23303, 1:3,000). The membranes were then visualized by enhanced chemiluminescence (ECL Kit, Servicebio technology, #G2014) and exposed to X-ray films.

Cultured cells or heart tissues were used to extract total protein with RIPA lysis buffer (Millipore) with protease inhibitor cocktail (Biomake). Next, standard Western blot was performed to determine the levels of interested proteins according to a protocol modified from previous studies [31 (link), 32 (link)]. The primary antibodies used for Western blot assay are :
Anti-IDO1 antibody (Abcam; 1:1000), anti-GAPDH antibody (Proteintech; 1:2000), anti-pAKT antibody (Cell Signaling Technology; 1:1000), anti-AKT antibody (Cell Signaling Technology; 1:1000), anti-pmTOR antibody (Cell Signaling Technology; 1:1000), anti-mTOR antibody (Cell Signaling Technology; 1:1000), anti-pS6K1 antibody (Cell Signaling Technology; 1:1000), anti-S6K1 antibody (Cell Signaling Technology; 1:1000). The secondary antibodies (1:5000) and ECL kit were purchased from Servicebio.

Cells collected from OS and hFOB1.19 were centrifuged and washed in PBS. Proteins were quantified with Bradford assays (Bio-Rad Laboratories) upon lysis of the cells with ice-cold RIPA buffer (Servicebio G2002). The proteins (30 ug) were extracted using a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The polyvinylidene fluoride (PVDF) membrane (Servicebio G6015-0.45) was then used to transfer the samples. At room temperature, 5% skim milk (Servicebio G5002) was added to block the membrane for 1 h. After overnight incubation at 4 °C, the specific antibody was washed with TBST (1 × TBS with Tween, Servicebio WGT8220) three times. After incubation for 30 min at room temperature, the secondary antibody was added. We washed the membrane with TBST three times after incubation with the secondary antibody. The protein bands were spotted with the aid of an ECL kit (Servicebio G2014). To semiquantify the relative band intensities, ImagePro Plus 6.0 (Media Cybernetics, Inc.) was employed. Normalization was performed using GAPDH as an internal reference gene.

Total protein was extracted from cells and tissues using radio-immunoprecipitation assay (RIPA) buffer (Servicebio Technology, Wuhan, China). A bicinchoninic acid (BCA) kit (Servicebio Technology, Wuhan, China) was used to quantify the protein concentration. Then total protein was separated by electrophoresis and transferred to a membrane. After blocking and washed three times with tris-buffered saline in tween (TBST), the membrane was incubated with primary antibodies overnight at 4 °C. Subsequently, the membrane was washed and incubated with the secondary antibodies. Finally, bands were visualized using an enhanced chemiluminescence (ECL) kit (Servicebio, Wuhan, China).

Briefly, proteins were extracted from osteosarcoma samples and cells using RIPA lysis buffer (Cell Signaling Technology, Danvers, MA, United States), and the protein concentration was determined using the BCA protein assay. Proteins were separated by 10% SDS-PAGE gel electrophoresis and then transferred onto PVDF membranes. After blocking with 5% skimmed milk, membranes were incubated with specific antibodies at 4°C overnight. The next day, membranes were washed twice with TBST and incubated with horseradish peroxidase-linked secondary antibodies (1:10,000) at room temperature for 1 h. Blots were visualized using the ECL Kit (Servicebio, Wuhan, China) and analyzed with ImageJ software. Antibodies targeting GAPDH, Ki67, E-cadherin, N-cadherin, and vimentin were purchased from Cell Signaling Technology, and antibodies targeting FHIT were purchased from Abcam (Cambridge, United Kingdom).