Validation of the specificity of the mGluR6 band obtained with this antibody, using retina lysates from WT and nob3 mice, which do not express mGluR6 (Maddox et al., 2008), is usually shown in Figure 7Bc-Myc Tetradecanoylcarnitine clone 9E10 hybridomas were obtained from the Developmental Studies Hybridoma Bank (The University of Iowa), and antibody was purified as described for TRPM1 antibodies and diluted to 1 1 g/ml for westerns. to ON bipolar cells (ON-BPC) is usually mediated by a post-synaptic G protein-coupled transduction cascade. Following deactivation of the glutamate receptor mGluR6 in response to light (Nakajima et al., 1993; Masu et al., 1995; Dryja et al., 2005; Zeitz et al., 2005), the TRPM1 ion channel subunit is required for depolarization (Audo et al., 2009; Li et al., 2009; Morgans et al., 2009; van Genderen et al., 2009; Shen et al., 2009). In addition to mGluR6 and TRPM1, the leucine-rich repeat proteins nyctalopin (Bech-Hansen et al., 2000; Pusch et al., 2000; Gregg et al., 2003) and LRIT3 (Zeitz et al., 2013; Neuill et al., 2014, 2015), the orphan GPCR GPR179 (Audo et al., 2012; Peachey et al., 2012; Ray et al., 2014), G proteins Gao (Dhingra et al., 2000, 2002), G3 (Dhingra et al., 2012), and G5 (Chen et al., 2003; Rao et al., 2007), and GTPase accelerating protein RGS7 or RGS11 (Mojumder et al., 2009; Shim et al., 2012; Cao et al., 2012), are also required for normal ON-BPC function. The roles of some of these proteins, and the interactions among the cascade components, remain poorly understood. TRPM1 has Tetradecanoylcarnitine been reported to interact with nyctalopin (Cao et al., 2011; Pearring et al., 2011) and GPR179 (Orlandi et al., 2013; Ray et al., 2014), both of which also interact with mGluR6 (Cao et al., 2011; Orlandi et al., 2013), suggesting the formation of a large multi-protein signaling complex. However, unlike these other cascade components, which are localized specifically to the bipolar cell dendritic tips (Nomura et al., Tetradecanoylcarnitine 1994; Masu et al., 1995; Morgans et al., 2006; Gregg et al., 2007; Peachey et al., 2012; Orlandi et al., 2013), TRPM1 is usually localized in bipolar cell bodies, dendrites, and axons (Morgans et al., 2009; Koike et al., 2010). This widespread distribution means that there is a large pool of TRPM1 that does not participate in the synaptic complex. In this study, we report TRPM1 monoclonal antibodies (mAbs) that Tetradecanoylcarnitine preferentially detect the non-synaptic pool of TRPM1, and identify the mAb epitope, which is likely masked by synapse-specific protein-protein interactions in the synaptic complex. Materials and methods Animals WT C57BL/6 mice were purchased from the Baylor College of Medicine Center for Comparative Medicine. Trpm1 knock-out mice (Trpm1tm1Lex; Lexicon Pharmaceuticals) were obtained from the European Mutant Mouse Archive and back-crossed to C57BL/6 for 15 generations before use (Agosto et al., 2014). Nob3 mice (Grm6nob3) (Maddox et al., 2008) were obtained from The Jackson Laboratory. Absence of the Pde6brd1 allele was confirmed by genotyping as described (Gimenez & Montoliu, 2001), and absence of the Crb1rd8 allele GDF2 (Mattapallil et al., 2012) was confirmed by PCR amplifying and sequencing the surrounding region of genomic DNA. All procedures were approved by the Baylor College of Medicine Animal Care and Use Committee. Cells clone 9 (Sf9) cells were maintained in InsectXpress medium (Lonza) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma) and 50 units/ml each of penicillin and streptomycin. Sf9 cells grown on coverslips in 24-well plates were infected with 50 l passage 3 baculovirus. Human embryonic kidney 293 (HEK) cells (ATCC) were maintained in Dulbeccos modified Eagle medium (Corning) supplemented with 10% FBS (Hyclone or Sigma). HEK cells in 6-well plates were transfected with 1C3 g plasmid DNA using Lipofectamine 2000 (ThermoFisher) according to the manufacturers instructions. Primary antibodies Full-length mouse mGluR6 was purified and complexed with amphipol as described for TRPM1 (Agosto et al., 2014), except protein and amphipol were mixed in a 1:3 (w/w) ratio. Mice were immunized with protein/amphipol complexes at the Baylor College of Medicine Monoclonal Antibody/Recombinant Protein Expression Core facility. Resulting mAbs were purified as described for TRPM1 antibodies, and clone 312 was used in westerns at 1C2 g/ml. In some experiments, the intermediate polyclonal mouse serum was used for westerns, diluted 1:1000. Validation of the specificity of the mGluR6 band obtained with this antibody, using retina lysates from WT and nob3 mice, which do not express mGluR6 (Maddox et al., 2008), is usually shown in Physique 7Bc-Myc clone 9E10 hybridomas were obtained from the Developmental Studies Hybridoma Bank.