Ab108337

A10 cells were transfected with siRNA or plasmid, and 1 day later, 2 mM Pi was treated for 3 days. On the following day, cells were collected by trypsinization, and protein was extracted using ice-cold RIPA buffer (Biosesang). Protein content was quantified using the Pierce BCA Assay Kit (Thermo Fisher Scientific), and 20 μg protein was separated on 10% SDS-polyacrylamide gel. The protein was transferred onto methanol-activated polyvinylidene difluoride (PVDF) membrane (Millipore) and blocked with 5% skim milk (BD Biosciences) in 1× TBS-T (Tris-buffered saline, 0.1% Tween 20). Membrane was incubated with primary antibody overnight at 4°C. Then after washing with 1× TBS-T three times, it was incubated with secondary antibody for 1 h at room temperature. The blot was visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and Fusion Fx (Vilber). The primary antibody used was anti-Alp antibody (ab108337, Abcam) and anti-Actin antibody (a2066, Sigma-Aldrich) with a 1:1,000 dilution, and the secondary antibody used was anti-rabbit immunoglobulin G (IgG), horseradish peroxidase (HRP)-linked antibody (7074, Cell Signaling Technology) with a 1:5,000 dilution. Results of western blots were analyzed by using ImageJ (https://imagej.nih.gov/ij/),⁴⁰ (link) and the expression of Alp was normalized by the expression level of a control gene, Actin.