|Detection of Rat and Mouse GPR64 by Western Blot. Western blot shows lysates of rat epididymis tissue and mouse epididymis tissue. PVDF membrane was probed with 2 µg/mL of Sheep Anti-Human/Mouse/Rat GPR64 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF7977) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for GPR64 at approximately 150 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.|
|GPR64 in Human Epididymus. GPR64 was detected in immersion fixed paraffin-embedded sections of human epididymus using Sheep Anti-Human/Mouse/Rat GPR64 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF7977) at 3 µg/mL overnight at 4 °C. Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained using the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS019) and counterstained with hematoxylin (blue). Specific staining was localized to plasma membranes of epithelial cells. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.|
GPR64 (G-Protein coupled Receptor 64; also HE6) is a 110 kDa (predicted) member of the B class of GCPRs. Within this class GPR64 belongs to a Large N-termini family-B 7-transmembrane (LNB-7TM) subclass of receptors (also known as adhesion-GCPRs). GPR64 has restricted expression, being found in stereocilia cell membranes of epididymal caput epithelial cells and, to a limited extent, on osteoblasts. The function of GPR64 is somewhat unclear, but in the epididymis, it may be involved in fluid transport. Mature human GPR64 is a 980 amino acid (aa) 7-TM glycoprotein (SwissProt Q8IZP9). It contains an extended extracellular N-teminus (aa 38-627), seven consecutive TM segments (aa 628-878) and a C-terminal cytoplasmic tail (aa 879-1010). The extended extracellular region possesses a juxtamembrane GPS domain (aa 567-618) that serves as a proteolytic cleavage site. Enzymatic activity here generates a 180 kDa soluble form that stays associated with a 40 kDa membrane-embedded fragment. Notably, isolation of the membrane fragment gives rise to oligomers that run at > 250 kDa in SDS-PAGE. There are multiple splice variants. The one used for immunization to generate this antibody contains a deletion of aa 88-101 (RefSeq NP_001073328). Four other splice forms show single block deletions of aa 65-67, 51-66, 52-75, and 906-956, respectively. Three others possesses aa substitutions; a 20 aa block for aa 52-101, and a common 12 aa block that can substitute for either aa 68-101 or aa 52-101. Over aa 38-64 and 68-553 of RefSeq NP_001073328, human and mouse GPR64 share 69% aa sequence identity.
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