Computational protocol: Enhanced Proliferation of Porcine Bone Marrow Mesenchymal Stem Cells Induced by Extracellular Calcium is Associated with the Activation of the Calcium-Sensing Receptor and ERK Signaling Pathway

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Protocol publication

[…] At the end of incubation, the pBMSCs were harvested and washed twice with PBS. Then Western blot was conducted as previously described []. Cells were lysed by lysis buffer, and the cell lysates were centrifuged to remove insoluble materials and the protein concentration of each sample was measured. Equal protein amounts of each sample were separated by SDS-PAGE and electroblotted to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk in TBST for 2 h at room temperature and then incubated with different primary antibodies, including anti-CaSR (1 : 500), anti-cyclin D1 (1 : 2000), anti-p21 (1 : 2000), anti-ERK1/2 (1 : 2000), and anti-phospho-ERK1/2 (1 : 2000), at 4°C overnight. Then the membranes were washed and incubated with different HRP-labeled secondary antibodies at room temperature for 1 h. Finally, the proteins were detected using the enhanced chemiluminescence detection reagents (Beyotime Institute of Biotechnology, Jiangsu, China) with a FluorChem M Fluorescent Imaging System (ProteinSimple, Santa Clara, CA, USA). Protein expressions were analyzed using ImageJ software. [...] All data are presented as means ± standard error of the mean (SEM). Statistical analysis was performed using SigmaPlot 12.5 (Systat Software, Inc., San Jose, CA). Differences between means were determined using Student's t-test and a confidence level of P < 0.05 was considered to be statistically significant. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Chemicals Calcium