Endolysosomal localization of TLR3 in cultured hepatocytes.
A, immunoblotting of indicated organelle markers, TLR3 and GRP78, in unfractionated cytoplasm of Huh7.5 cells, 7.5-TLR3 cells, and 7.5-TLR3-derived shG78-3 cells with stable knockdown of GRP78 (left), and in sucrose density gradient centrifugation-separated fractions of 7.5-TLR3 cells (middle), and shG78-3 cells (right). B, confocal fluorescence microscopy of immunostained 7.5-TLR3 cells (upper panels) and shG78-13 cells with stable GRP78 knockdown (lower panels) demonstrating co-localization of TLR3 (green fluorescence) and LAMP2 (red fluorescence). Nuclei were counterstained with DAPI (blue fluorescence). C, confocal fluorescence microscopy illustrating partial co-localization of TLR3 (red fluorescence) and calreticulin (green fluorescence) in immunostained 7.5-TLR3 cells. D, no TLR3 immunolabeling was detected in Huh7.5 cells. E, confocal fluorescence microscopy showing co-localization of TLR3 (green fluorescence) and LAMP2 (red fluorescence) in primary mouse hepatocytes. Nuclei were counterstained with DAPI (blue fluorescence).