Bca protein colorimetric assay kit

Cell lysis buffer for Western and IP (P0013, Beyotime) was used to lyse HGC27 and AGS cells after they had been exposed to various doses of NTP for 24 h. The BCA protein Colorimetric Assay Kit (E-BC-K318-M, Elabscience) was subsequently utilized to determine the total protein. After the protein was separated by electrophoresis, the membrane was transferred using polyvinylidene difluoride (PVDF) at an ice bath temperature. Five percent skim milk was used to block the portions. In a 4 °C shaker, the matching antibodies were incubated overnight. After rinsing with Tris-buffered saline and Tween (TBST), the film was treated with secondary antibodies (S0002, Affinity) for 1.5 h at room temperature. Finally, the imaging system was used to build the improved ECL chemiluminescent substrate kit (36222, Yeasen). For some of the PVDF membranes, Western blot fast stripping buffer (PS107, Epizyme) was used. After completing Western blot luminescence detection, the PVDF membranes were rinsed with TBST for 5 min. Add 10 mL of fast stripping buffer to cover the membrane and rinse for 20 min. The stripping solution was removed, and TBST was added to rinse the PVDF membrane for 5 min. The membrane was sealed with skim milk powder for 2 h, the PVDF membrane was washed with TBST, and then the other antibodies were added again. The gray value was analyzed and calculated using ImageJ software.

To investigate osteogenic differentiation, BMSCs were cultured on DE-FS (n = 6 each group) loaded with or without exosomes supplemented with osteogenic media for 2 weeks. Then, the cells underwent 2 times of PBS washing, followed by 4% paraformaldehyde fixation for 5 min, and incubation with 2% Alizarin Red S (ARS) solution (Sigma) for 30 min. Finally imaged using a microscope (Nikon, Tokyo, Japan). To quantify the calcium amount on the DE-FS, the red matrix precipitate was solubilized in 10% cetylpyridinium chloride (Sigma). The eluted stain was read at 562 nm. In addition, alkaline phosphatase (ALP) activity of BMSCs that cultured on DE-FS (n = 6) loaded with or without exosomes undergo differentiation by incubation in osteogenic medium for 7 days was detected using an ALP Activity Assay Kit (Elabscience, Wuhan, China). The wavelength of 520 nm was set for detection in microplate reader. The data was normalized to the total protein contents determined by a BCA Protein Colorimetric Assay Kit (Elabscience).

BCA Protein Colorimetric Assay Kit (Elabscience Biotechnology Co., Ltd., China) was used to quantify the total protein of brain cortex tissues and SY5Y cells. Samples with an equal amount of protein were loaded and separated via sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The proteins were transferred to the appropriate polyvinylidene fluoride membrane (Millipore, USA), blocked with 5% skimmed milk for 1 h, and thens incubated with diluted antibodies at 4°C overnight. For the in vivo experiments, Drp1 (1:2000), Fis1 (1:1500), MFF (1:8000), OPA1 (1:2000), Mfn1 (1:1000), Mfn2 (1:2000), and β‐actin (1:5000) antibodies were purchased from Proteintech Group, Inc., China. Meanwhile, for the in vitro experiments, the antibodies included Drp1 (1:800; AiFang Biological, China), Fis1 (1:1500; Bioss, China), OPA1 (1:1000; AiFang Biological, China), Mfn1 (1:1000; Affinity, China), Mfn2 (1:500; AiFang Biological, China), and SENP6 (1:1000; Abcam, USA). Furthermore, MFF (1:2000), SUMO1 (1:2000), SUMO2/3 (1:500), SENP1 (1:2000), SENP2 (1:1000), SENP3 (1:800), SENP5 (1:3000), and β‐actin (1:5000) were obtained from Proteintech Group, Inc., China. Then, the membranes were washed and incubated with secondary antibodies for 2 h at room temperature. After rewashing with TBST buffer, the membranes were added ECL reagent to enable the detection of the protein bands.