Cell lysis buffer for western and ip without inhibitors

The detected cells were lysed by Cell lysis buffer for Western and IP without inhibitors (Beyotime Biotechnology, China), and the supernatant was collected for semi‐quantitative analysis of ALP using an Alkaline Phosphatase Assay Kit (Beyotime Biotechnology, China) according to the manufacturers’ instructions. Para‐nitrophenol (p‐nitrophenol), as a common substrate for phosphatase activity, can produce yellow products under alkaline conditions. Optical density (OD) values were measured using a microplate reader (SpectraMax Plus384, Molecular Devices, USA) at 405 nm. The expression level of ALP was standardized to the total protein content of cells to obtain the absorbance index.

ALP quantification and staining were used to characterize the ALP activity of ECM-secreting BMSCs after 7 days of incubation (Yan et al., 2021 (link)). ALP quantification was measured according to the instructions of the ALP assay kit (Beyotime, China). BMSCs were seeded at 5 × 10⁴ cells/well on the MCs (2 mg/well) and M-ICA (2 mg/well) in 48-well plates. After incubation for 7 days, the samples were rinsed with PBS and lysed by adding 200 μl cell lysis buffer for Western and IP without inhibitors (Beyotime, China). The mixed lysing solutions were frozen at −80°C for 30 min, thawed at 37°C and centrifuged for 15 min at 4°C, 13,000 rpm. The p-nitrophenol phosphate (pNPP) assay and bicinchoninic acid (BCA) solution were added and incubated at 37°C for 30 min away from light. The OD405/OD562 ratio was read as the ALP quantitative evaluation. For ALP staining, the cell-cultured microcarriers were fixed by 4% PFA for 20 min and rinsed three times with PBS. Then ALP staining assay was performed according to the ALP staining kit (Beyotime, China) protocol. After 24 hof incubation away from light, the samples were observed with a stereomicroscope.

Co-Immunoprecipitation (Co-IP) of Sirt3 with ubiquitin in Atg7^+/+ and Atg7^−/− K562 cells. Cells were splited using cell lysis buffer for Western and IP without inhibitors (Beyotime, Nantong, China). Proteins were incubated with Sirt3 antibody (Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C, then incubated with Protein A+G Agarose (Beyotime, Nantong, China) for 2 h at 4°C. The purified proteins were detected with western blotting. Left panel: western blotting results of the input for whole cell lysate used as a positive control; right panel: Co-IP between Sirt3 and ubiquitin. IgG (Cell Signaling Technology, Danvers, MA, USA) used as a negative control. The lower panels are western blotting results of Sirt3.

HA-VSMC cells or transfected HA-VSMC cells were cultured with 30 mmol/L glucose for 14 days. Then, cells were washed, scraped into a solution, and lysed with Cell lysis buffer for Western and IP without inhibitors (Beyotime Biotechnology, Shanghai, China). Cell lysates were measured for alkaline phosphatase (ALP) activity using an Alkaline Phosphatase Assay Kit (Beyotime Biotechnology, Shanghai, China) and for osteocalcin secretion using an osteocalcin assay kit (Jiancheng, Nanjing). ALP activity and osteocalcin secretion were normalized to the total cellular protein of the cellular layers and then normalized to that of the control [21 (link)].