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. 2014 Oct 30;4:6836. doi: 10.1038/srep06836

Figure 5. Localization of SLC18B1 protein in cultured astrocytes.

Figure 5

(a) Western blotting with purified antibodies indicated the presence of rat SLC18B1 protein in the membrane of hippocampal astrocytes (40 μg protein) (left). The absorbed antibodies were used as a control (right). The position of SLC18B1 protein is marked by an arrow. (b) Indirect immunofluorescence microscopy revealed that SLC18B1 was expressed in primary cultured rat astrocytes. Inset, control staining with normal serum. Bar  =  10 μm. (c) SLC18B1 immunofluorescence was present in the vesicular component of cultured hippocampal astrocytes. Astrocytes were double immunostained with sets of antibodies against SLC18B1 and VAMP2, CgA, GM130, EEA1 or PDI. Bar  =  10 μm. VAMP2 and CgA (red) were used as vesicular markers. (d) Sucrose density gradient analysis of SLC18B1 localization. The astrocyte membrane fraction was subjected to continuous sucrose density gradient centrifugation, and fractionated into 10 fractions from the bottom. Each fraction (50 μL) was solubilized with SDS sample buffer and immunoblotted with antibodies against SLC18B1, VAMP2, secretogranin II, chromogranin A (CgA), V-ATPase subunit A, and cathepsin D.