Confirmation of CaSR expression in HAoECs through immunostaining and western immunoblotting. [A (i)] Immunostaining of the HAoEC showing the expression and localization of the CaSR protein with the anti-CaSR antibody (LS-C417520, green), (ii) DAPI staining of the nucleus, (iii) immunostaining of HAoEC membrane protein, VE-cadherin (red), and (iv) merged image of the cell confirming cytosolic localization of the CaSR protein. The anti-CaSR antibody (LS-C417520) targets endogenous CaSR. (B) Western immunoblot showing CaSR protein expression in the cytoplasmic protein fraction of HAoECs at ~130 kD but a much smaller molecular weight protein (~40 kD) in the membranal fraction of HAoECs. The purity of the membrane and cytoplasmic fractions were confirmed by the presence of VE-cadherin (in the membranal fraction) and β-actin (in the cytoplasmic fraction).