Specificity of the WDR81 antibody for the antigen.

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Specificity of the WDR81 antibody for the antigen. Specificity of the WDR81 antibody for the antigen. Preincubation of the WDR81 antibody with the immunization (i.m.) peptide abolished the detection of WDR81 protein on Western blots of brain and spinal cord extracts (A), as well as in immunostained (red) Purkinje cell neurons of the cerebellum (B), in brainstem neurons (B), and in the retina cell layers (B; IS, OPL, INL, IPL, GCL) of adult wild-type mice. Retinal cell nuclei were visualized by DAPI counterstaining (blue). In control experiments, the detection of WDR81 protein is not affected by the preincubation of the WDR81 antibody in 1× PBS. C, The WDR81 antibody recognizes a band at ∼100 kDa in protein extracts of HEK cells transfected with a plasmid that encodes the isoform 2 of the mouse WDR81 protein, which is absent in mock-transfected (negative control) and GFP-transfected HEK cells. The presence of the glycosylation inhibitor tunicamycin in the cell medium does not reduce the molecular weight of the protein. WDR81 is detected at lower molecular weights (90 and 80 kDa) in P21 cerebellar extracts. Actin was used for the normalization of protein levels, and thymus was used as negative control tissue in the Western blots shown in A. OS, Outer segment layer; IS, inner segment layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars: B (cerebellum and brainstem), 50 μm; B (retina), 75 μm. Maria Traka et al. J. Neurosci. 2013;33:6834-6844 ©2013 by Society for Neuroscience