Protein quantification kit

Western blotting was performed as previously described [20 ]. Proteins were collected using RIPA buffer (P0013B, Beyotime, China), protease inhibitors (p1005, Beyotime, China), and phenylmethylsulfonyl fluoride (ST505, Beyotime, China), quantified by a protein quantification kit (P0011, Beyotime, China), and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, electrophoresis was conducted to transfer the proteins into a PVDF membrane (160-0184, Bio-Rad, USA). The membrane was blocked by 5% dried skimmed milk powder for 1 h and then incubated with antibodies against Akt (1: 500, 56kDa, ab8805, Abcam, UK), phosphorylated (p)-Akt (1: 1000, 56kDa, ab38449, Abcam, UK), endothelial nitric oxide synthase (eNOS) (1: 1000, 133kDa, ab76198, Abcam, UK), p-eNOS (1: 500, 140kDa, ab76199, Abcam, UK), and GAPDH (1: 1000, 36kDa, ab8245, Abcam, UK). After incubation for 24 h, the membrane was washed by 1% Tris-buffered saline with Tween 20 and incubated with goat anti-rabbit secondary antibody (1: 10 000, ab205718, Abcam, UK) or goat anti-mouse secondary antibody (1: 10000, ab6789, Abcam, UK). An ECL luminescence kit (PE0010, Solarbio, China) and a gel imaging system (FluorChem FC3, Alpha, USA) were used to expose the membrane and visualize the protein bands, respectively. ImageJ2x (Rawak Software, Germany) was used to analyze the results.

Cells were cultured for 3 days and 7 days in osteogenic induction medium. The osteogenic induction medium contained growth medium with 50 μg/ml l-ascorbic acid (Sigma) and 10 mM β-glycerophosphate (Sigma) and 0.1 μM dexamethasone (Sigma). A commercially available ALP staining kit (Beyotime) was used to stain ALP according to manufacturer's instructions. ALP quantification kit (Beyotime) and protein quantification kit (Beyotime) were used to measure the activity of ALP and total protein according to manufacturer's instructions [38 (link)].

ADH4 protein expression of the resected tissues was examined using western blotting. The tissues were first treated using a lysis solution (RIPA, Radio-Immunoprecipitation Assay buffer; Thermo Fisher Scientific, USA), which contained 1% (v/v) protease inhibitors (cat. no. P8340; Merck KGaA) to extract protein samples. The protein concentration was measured and normalized using Protein Quantification kit (BCA Assay; Beyotime, China). Western blot analysis was then performed as previously described (18 (link)). The membranes were blocked in Tris-Buffer Saline Tween 20 (1×TBST) containing 5% non-fat milk at room temperature for 45 min. Then, membranes would be incubated with anti-ADH4 primary antibody (cat. no. Ab137077; 1:1,000 dilution; Abcam) overnight at 4°C and HRP-conjugated goat anti-rabbit secondary antibody (cat. no. Ab6721; 1:1,000 dilution; Abcam) for 1 h at room temperature. Enhanced chemiluminescence reagent (SuperSignal™ West Atto Ultimate Sensitivity Substrate; Thermo Fisher Scientific, Inc.) was used for immunodetection. The level of actin protein expression was measured as an internal standard (anti-β actin antibody; cat. no. Ab8227; 1:1,000 dilution; Abcam). The density of protein band was quantitatively measured using Image J software (v1.52a, USA).

Cells were harvested, washed three times with pre-cold 1× PBS, resuspended in 5 ml of pre-cold mitochondrial isolation buffer (225 mM mannitol, 75 mM sucrose, 30 mM Tris–HCl (pH 7.4), 1 mM PMSF), and the cell membranes ruptured by using a manual homogenizer (T10 Basic ULTRA-TURRAX® (IKA)) for 15 min at 4°C. The homogenized sample was centrifuged at 600 g at 4°C for 5 min to remove cell debris. The supernatant was collected and further centrifuged at 8000 g at 4°C for 10 min. The pellet (mitochondria) was stored at –80°C for further use.
The isolated mitochondria were resuspended in solubilization buffer (50 mM NaCl, 50 mM imidazole, 2 mM 6-aminohexanoic acid and 1 mM EDTA). The protein concentration was determined using a Protein Quantification Kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. Following standard procedure, 500 μg mitochondria were lysed by 20% digitonin (mitochondria: digitonin = 1:8, m/m) at 4°C for 10 min. 30 μg of total mitochondrial proteins were separated on a 3–12% Bis–Tris native PAGE gel at 4°C overnight and then processed using the same procedure described in the western blot or stained with coomassie brilliant blue.