HEK293 was originally purchased from American Type Culture Collection (Manassas, VA, USA). Mouse embryonic fibroblasts (MEFs) were extracted from NIH mouse embryos as report [23 (link)]. Cells were cultured using Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 μg/ml), and at 37° C in 5% CO2. Rapamycin (HY-10219) and Stat-3 specific inhibitor (HO-3837, HY-100453) were ordered from MedChemexpress (Monmouth Junction, NJ, USA). Primary antibodies against Runx-2 (sc-390351), OPN (sc-21742), p-Akt1/2/3 (sc-377556), Akt1/2/3 (sc-56878), p-mTOR (sc-517464), and mTOR (sc-293089) were ordered from Santa Cruz Biotechnology (Santa Cruz, CA, USA); primary antibodies against IL-6 (21865-1-AP), Rictor (27248-1-AP), and Raptor (20984-1-AP) were ordered from Proteintech (Wuhan, China); primary antibody against β-actin (AC026) was ordered from ABclonal technology (Wuhan, China).
Sc 390351
Manufactured by Santa Cruz Biotechnology
Sourced in United States, China
Sc-390351 is a laboratory reagent manufactured by Santa Cruz Biotechnology. It is a chemical compound used for research purposes. The core function of this product is to serve as a research tool, without further interpretation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Ab209484, by Abcam (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Pm036, by MBL Life Science (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Sc 21742, by Santa Cruz Biotechnology (1 mentions)
Rapamycin hy 10219, by MedChemExpress (1 mentions)
Sc 377556, by Santa Cruz Biotechnology (1 mentions)
Sc 56878, by Santa Cruz Biotechnology (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Ab14106, by Abcam (1 mentions)
Ab14933, by Abcam (1 mentions)
D4k9n, by Abcam (1 mentions)
Sc 390991, by Santa Cruz Biotechnology (1 mentions)
Ecl system, by Merck Group (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Supersignal west pico trial kit, by Thermo Fisher Scientific (1 mentions)
Sc 292838, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Ripa solution, by Beyotime (1 mentions)
13 protocols using sc 390351
1
Osteogenic Differentiation of HEK293 and MEFs
2
Protein Expression Analysis in Aortic Tissue
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The aorta tissue was homogenized and centrifuged at 13,000 rpm for 30 min at 4 °C to take the supernatant. The extract of aorta tissues was prepared in lysis buffer [20 mM Tris-HCl, 1 mM dithiothreitol (DTT), 5 mM EGTA, 0.5 mM PMSF, 20 μM leupeptin, and 20 μM aprotonin]. Separation of total protein were done by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10–14% acrylamide gels, then transferred to a polyvinylidine diflouride (PVDF). After blocking with 5% fat-free milk for 1 hour, the membranes were incubated overnight at 4 °C with the respective primary antibodies rabbit antibodies against SM22α (1:1000, ab14106 Abcam, Cambridge, MA, USA), α-SMA (1:1000, D4K9N, USA), BMP-2 (1:500, ab14933, Abcam, Cambridge, MA, USA), Runx2 (1:100, sc-390351, Santa Cruz, Dallas, TX, USA), Nrf2 (1:500, tcba 1560, Taiwan), Keap1 (1:1000, 10503-2-AP, USA), and HO-1 (1:250, sc-390991, Santa Cruz, Dallas, TX, USA). The blots were visualized with enhanced chemiluminescence (ECL) system (Millipore Corporation, Billerica, MA 01821 USA) and Image J software was used to scan and quantify the gray values.
3
Osteogenic Protein Expression under Varying Oxygen Conditions
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Protein expression of ERK1/2, p-ERK1/2 and osteogenic maker (RUNX2) was analyzed by Western blotting assay. Briefly, after total protein of rTDSCs osteogenic-cultured in different oxygen tension conditions for 21 days was extracted with RIPA solution (Beyotime, China), protein samples were subjected to SDS-PAGE and transferred to PVDF membrane (Roche). Then, the PVDF membrane was blocked with 5% bovine serum albumin (BSA) and incubated with primary antibodies (ERK1/2, 1:500, sc-292838, Santa Cruz; p-ERK1/2, 1:500, sc-101761, Santa Cruz; RUNX2, 1:500, sc-390351, Santa Cruz; β-actin, 1:1000, 60008-1-Ig, Proteintech) overnight at 4°C and HRP-conjugated secondary antibodies (ZSGB-BIO, China) for 2 hours at room temperature. Finally, protein bands on the PVDF membrane were visualized using the SuperSignal West Pico Trial Kit (Thermo) and analyzed using the Image J software (National Institutes of Health, USA).
4
Western Blot Analysis of Protein Expression
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Protein expression in the samples was analysed by western blotting. The total protein lysate was extracted with cell lysis buffer for western blotting and immunoprecipitation (No. P0013, Beyotime) and denatured by boiling. Protein samples were resolved on 12 % SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (PVDF Western Blotting Membranes, Roche). Membranes were blocked in PBS containing 5 % (w/v) non-fat dry milk and 0.1 % Tween-20 for 2 h, incubated with the appropriate antibodies overnight, washed with TBST buffer, and then incubated with secondary antibodies for 2 h. Blots were developed using a horseradish peroxidase-linked secondary antibody and developed with a chemiluminescent detection system (Phototope-HRP Western blot detection kit, New England Biolab).
The primary antibodies used for blotting were as follows: anti-Smad5 (1:1000, 12534, Cell Signaling Technology), anti-p-Smad5 (1:1000, 9516S, Cell Signaling Technology), anti-OGN (1:1000, sc-374463, Santa Cruz), anti-Runx2 (1:1000, sc-390351; Santa Cruz), and anti-β-actin (1:1000, sc-47778, Santa Cruz).
The primary antibodies used for blotting were as follows: anti-Smad5 (1:1000, 12534, Cell Signaling Technology), anti-p-Smad5 (1:1000, 9516S, Cell Signaling Technology), anti-OGN (1:1000, sc-374463, Santa Cruz), anti-Runx2 (1:1000, sc-390351; Santa Cruz), and anti-β-actin (1:1000, sc-47778, Santa Cruz).
5
Immunohistochemical Analysis of Bone Remodeling Markers
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Sections were dewaxed in xylene and then rehydrated through an ethanol concentration gradient. Tissue samples were then deactivated with 3% H 2 O 2 . Antigen retrieval was performed using trypsin (Cat. #T8150, Solarbio, Beijing, China), followed by blocking the sections with 5% goat serum (Cat. #AR0009, BOSTER, Pleasanton, CA, USA) for 1 hour. Subsequently, sections were incubated overnight at 4 °C with specific antibodies against βcatenin (1∶500, Cat. #PK02151, Abmart, Shanghai, China), RUNX2 (1∶200, Cat. #sc-390 351, Santa Cruz Biotechnology, Dallas, TX, USA), OSX (1∶500, Cat. #ab209484, Abcam, Cambridge, UK), OPG (1∶500, Cat. #DF6824, Affinity, Cincinnati, OH, USA), NFATc1 (1∶200, Cat. #sc-7294, Santa Cruz Biotechnology, Dallas, TX, USA), c-Fos (1∶500, Cat. #66590-1-Ig, Proteintech, Rosemont, IL, USA), CTSK (1∶200, Cat. #sc-48353, Santa Cruz Biotechnology, Dallas, TX, USA), and RANKL (1∶500, Cat. #bs-20647R, Bioss, Woburn, MA, USA). Afterward, the sections were incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour, followed by counterstaining with hematoxylin and sealing with neutral resin. Immunoreactivity was detected using an optical microscope (Olympus, Tokyo, Japan).
6
Immunofluorescence Staining for LC3 and Runx2
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Cells were seeded on glass cover slips in 24-well plates at a density of 3 × 10 6 cells/well. Cells were fixed with 4% PFA at 4°C and washed with PBS. The fixed cells were permeabilised with 0.1% triton X100 (Sigma) and blocked with 2% goat before incubating with primary antibodies LC3 (1:300; rabbit polyclonal; PM036; MBL International) or Runx2
(1:100; Mouse; Sc-390351; Santa Cruz Biotechnology) overnight at 4°C.
(1:100; Mouse; Sc-390351; Santa Cruz Biotechnology) overnight at 4°C.
7
Tissue Fixation and Histological Analysis
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Tissues were fixed in 10% NBF for 24 h before being dehydrated, embedded in paraffin wax, and sectioned (4 μm) using standard procedures as previously described (Zhu et al., 2016) . Sections were dewaxed in xylene and stained with Von Kossa and alizarin red (Sigma) to visualize phosphate and calcium deposition, respectively.
For immunofluorescence analysis, sections were demasked with 10 mM sodium citrate buffer. Endogenous peroxidase and nonspecific antibody binding were blocked before overnight incubation with primary antibodies LC3 (1:300; PM036; MBL International), Runx2 (1:200; Sc-390351; Santa Cruz Biotechnology), or ATG3
(1:300; ab108251; Abcam) at 4°C. The sections were then incubated for 1 h in the dark with Alexa Fluor@488 anti-rabbit antibody (Life Technologies; A11034) and Alexa Fluor@647 goat anti mouse antibody (Life Technologies; A21236). Sections were washed in PBS and stained with Hoescht (1:10,000; Sigma) and then mounted onto slides with Prolong ® Gold Anti-Fade Reagent (Life Technologies).
Fluorescence signal was detected under a Zeiss LSM 710 inverted confocal microscope. Control sections were incubated with nonimmune goat IgG (2 μg IgG/ml) in place of the primary antibody.
For immunofluorescence analysis, sections were demasked with 10 mM sodium citrate buffer. Endogenous peroxidase and nonspecific antibody binding were blocked before overnight incubation with primary antibodies LC3 (1:300; PM036; MBL International), Runx2 (1:200; Sc-390351; Santa Cruz Biotechnology), or ATG3
(1:300; ab108251; Abcam) at 4°C. The sections were then incubated for 1 h in the dark with Alexa Fluor@488 anti-rabbit antibody (Life Technologies; A11034) and Alexa Fluor@647 goat anti mouse antibody (Life Technologies; A21236). Sections were washed in PBS and stained with Hoescht (1:10,000; Sigma) and then mounted onto slides with Prolong ® Gold Anti-Fade Reagent (Life Technologies).
Fluorescence signal was detected under a Zeiss LSM 710 inverted confocal microscope. Control sections were incubated with nonimmune goat IgG (2 μg IgG/ml) in place of the primary antibody.
8
Histological Characterization of Meniscus Explants
9
Protein Expression Analysis in Cells
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In short, cells were washed with cold PBS three times and lysed with radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors for 30 min on ice. The total protein concentration was quantified by a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Samples were separated by SDS/polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes (Millipore). Then the membranes were blocked with 5% fat-free milk dissolved in TBST (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, and 0.05% Tween 20) at room temperature for 1 h and incubated with primary antibodies against GAPDH (1:1,000, CW0100M; CWBIO), NAT10 (1:1,000, 13365-1-AP; Proteintech), RUNX2 (1:1,000 dilution, sc-390351; Santa Cruz), and Osterix (1:1,000, ab209484; Abcam) overnight at 4°C. After that, the membranes were incubated with the appropriate secondary antibody, either horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:3,000; Beyotime) or anti-mouse IgG (1:3,000; Beyotime), at room temperature for 1 h and visualized with chemiluminescent HRP substrate (Millipore, Billerica, MA, USA). ImageJ software was used to analyze the band intensities.
10
Histological and Immunofluorescence Analysis of Embryonic Development
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For histology and immunofluorescence, the heads of the pups and embryos were fixed overnight in 4% PFA, dehydrated to 100% ethanol, embedded in paraffin, and sectioned into 5-μm-thick slices. Immunohistochemical and immunofluorescence staining was performed with anti-Hdac4 polyclonal antibody (ab12172, Abcam, United States, 1:250), anti-Runx2 antibody (sc-390351, Santa Cruz, United States, 1:50), anti-Ki67 rabbit monoclonal antibody (ab16667, Abcam, 1:200), anti-P21/Cdkn1a mouse monoclonal antibody (sc-6246, Santa Cruz, 1:50), goat polyclonal secondary antibody to rabbit IgG (ab150078, Abcam, 1:500), and goat polyclonal secondary antibody to mouse IgG (ab150114, Abcam, 1:500) following a previously described protocol (Zhao et al., 2020 (link)). Images were captured using an Olympus IX83 inverted microscope.
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