Fig. 4.

ALPL deficiency promoted the internalization of L-type Ca²⁺ channels via binding to α2δ subunits. a Representative images of confocal laser scanning microscopy showing a region of membrane colocalization for ALPL (DsRed) and Ca_V1.2 (FITC-labeled) or Ca_V1.3 (FITC-labeled) in WT BMSCs. No region of membrane colocalization was found in alpl^+/− BMSCs. Scale bar, 10 μm. b ALPL immunoprecipitated Ca_V1.2 and Ca_V1.3. The left lane shows the expression of Ca_V1.2 and Ca_V1.3, and the right lane shows the levels of Ca_V1.2 and Ca_V1.3 following immunoprecipitation with an anti-ALPL antibody. c Ca_V1.2 and Ca_V1.3 immunoprecipitated ALPL. The left panel shows the expression of ALPL, and the right panel shows the level of ALPL following immunoprecipitation with anti-Ca_V1.2 or anti-Ca_V1.3 antibodies. d Representative images of confocal laser scanning microscopy showing the membrane colocalization region of ALPL (Cy3-labeled) and Ca_V1.2 (FITC-labeled) or Ca_V1.3 (FITC-labeled) in alpl^−/− BMSCs overexpressing ALPL and the α2δ subunit. No membrane colocalization region was found in alpl^−/− BMSCs and alpl^−/− BMSCs overexpressing ALPL or the mutant α2δ subunit. Scale bar, 10 μm. e Western blot analysis showed membrane expression of Ca_V1.2 or Ca_V1.3 in alpl^−/− BMSCs overexpressing ALPL and the α2δ subunit. No membrane expression of Ca_V1.2 or Ca_V1.3 was found in alpl^−/− BMSCs and alpl^−/− BMSCs overexpressing ALPL or the mutant α2δ subunit. No significant change in cytoplasmic Ca_V1.2 or Ca_V1.3 was found in alpl^−/− BMSCs, alpl^−/− BMSCs overexpressing ALPL and the mutant α2δ subunit, or alpl^−/− BMSCs overexpressing ALPL and the α2δ subunit. f α2δ immunoprecipitated ALPL. The left panel shows the expression of ALPL, and the right panel shows the level of ALPL following immunoprecipitation with an anti-α2δ antibody in alp/^f/f and alpl^−/− BMSCs. β-actin was used as a protein loading control. The representative results from three independent experiments are shown