Anti magt1

Total proteins of BMMSCs were isolated using RIPA (Cell Signaling Technology, USA). The automated western blot was performed using Simple Wes (Protein Simple, USA) following the manufacturer’s protocol. Briefly, 2.5 μg of protein from the cell lysates was added to the standard fluorescent mastermix, then was loaded into corresponding wells of the prefilled Wes assay plate, along with antibody diluent (Protein Simple, USA), anti-MagT1 (Proteintech, USA), anti-DSP (Santa Cruz, USA), anti-DMP-1 (Genetex, USA), anti-ALP (Affinity, USA), anti-ERK (Cell Signaling Technology, USA), anti-p-ERK (Cell Signaling Technology, USA), anti-JNK (Cell Signaling Technology, USA), anti-p-JNK (Cell Signaling Technology, USA), anti-p38 (Cell Signaling Technology, USA), anti-p-p38(Cell Signaling Technology, USA), anti-β-Tublin (Affinity, USA), anti-rabbit secondary antibody (Protein Simple, USA), and Streptavidin-HRP, followed by luminal peroxide mix. The imaging and analysis were done with compass software (Protein Simple).

Cells were lysed in 50 mM TrisHCl pH 8.0 containing 150 mM NaCl, 1 mM EDTA, 1% NP-40. Cell extracts (100 μg/lane) were resolved by 8 or 10% SDS-PAGE, transferred to nitrocellulose sheets at 100 mA for 16 h, and probed with anti-TRPM7 (Bethyl), anti-MagT1 (ProteinTech), anti-PgP (ThermoScientific), and anti-actin (Sigma Aldrich) antibodies. Secondary antibodies were labelled with horseradish peroxidase (GE Healthcare). The SuperSignal chemiluminescence kit (Pierce) was used to detect immunoreactive proteins. All the results were reproduced at least three times and a representative Western blot is shown. Densitometric analysis was performed by the ImageJ software and ratio between the protein of interest and actin was calculated on three separate experiments (see Supplementary).