Abstract
Pannexin1 (Panx1) channels release cytosolic ATP in response to signaling pathways. Panx1 is highly expressed in the central nervous system. We used four antibodies with different Panx1 anti-peptide epitopes to analyze four regions of rat brain. These antibodies labeled the same bands in Western blots and had highly similar patterns of immunofluorescence in tissue culture cells expressing Panx1, but Western blots of brain lysates from Panx1 knockout and control mice showed different banding patterns. Localizations of Panx1 in brain slices were generated using automated wide field mosaic confocal microscopy for imaging large regions of interest while retaining maximum resolution for examining cell populations and compartments. We compared Panx1 expression over the cerebellum, hippocampus with adjacent cortex, thalamus, and olfactory bulb. While Panx1 localizes to the same neuronal cell types, subcellular localizations differ. Two antibodies with epitopes against the intracellular loop and one against the carboxy terminus preferentially labeled cell bodies, while an antibody raised against an N-terminal peptide highlighted neuronal processes more than cell bodies. These labeling patterns may be a reflection of different cellular and subcellular localizations of full-length and/or modified Panx1 channels where each antibody is highlighting unique or differentially accessible Panx1 populations. However, we cannot rule out that one or more of these antibodies have specificity issues. All data associated with experiments from these four antibodies are presented in a manner that allows them to be compared and our claims thoroughly evaluated, rather than eliminating results that were questionable. Each antibody is given a unique identifier through the NIF Antibody Registry that can be used to track usage of individual antibodies across papers and all image and metadata are made available in the public repository, the Cell Centered Database, for on-line viewing, and download.
Highlights
One mechanism of paracrine cell–cell communication occurs by ATP release and signal transduction through purinergic receptors (Burnstock, 2011)
Membrane bound purinergic receptors are found on the plasma membrane of neurons and non-neuronal cells such as astrocytes and microglia (Fields and Stevens, 2000)
Several studies have shown an association of the purinergic receptors with pannexin1 (Panx1), a connexin-like protein, which when stimulated acts as an ATP release channel (Locovei et al, 2006b; Nishida et al, 2008; Silverman et al, 2009; Kim and Kang, 2011; Poornima et al, 2011; Vessey et al, 2011)
Summary
One mechanism of paracrine cell–cell communication occurs by ATP release and signal transduction through purinergic receptors (Burnstock, 2011). ATP signaling stimulates neurotransmission, neuromodulation, and secretion as well as playing a role in cell proliferation, differentiation, and inflammation (Fields and Stevens, 2000). Several studies have shown an association of the purinergic receptors with pannexin (Panx1), a connexin-like protein, which when stimulated acts as an ATP release channel (Locovei et al, 2006b; Nishida et al, 2008; Silverman et al, 2009; Kim and Kang, 2011; Poornima et al, 2011; Vessey et al, 2011).
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