Differential expression of α-chimaerin: Identification by ddPCR, confirmation by northern blot and validation of antibodies. A. Portion of a ddPCR sequencing gel showing a ddPCR product (arrow) which is expressed more highly in dark reared (D5) than normal (N5) cat visual cortex at 5 weeks and more highly in normal (N20) than dark reared (D20) cat visual cortex at 20 weeks. Independently isolated RNA samples from two cats at each rearing/age condition were run together (8 lanes) to help eliminate false positives. B. Top. Northern blot confirming differential expression of the ddPCR fragment. Total cat visual cortical RNA from each of the four rearing/age conditions was loaded and probed with the cloned ddPCR fragment. Asterisks indicate 28s rRNA. Bottom. The same filter is shown after stripping and reprobing with GAPDH. C. Western blots indicated that each antibody recognized a single band of appropriate molecular weight (α1: 38 kD, α2: 53 kD). α1-Chimaerin (Top) was expressed about equally in mouse visual (V), frontal (F) cortex and cerebellum (C). α2-Chimaerin (Middle) was expressed highly in neocortical structures but at very low levels in cerebellum. Filter was stripped and reprobed with an antibody to actin (Bottom) as a control for loading errors.
Yang et al. BMC Neuroscience 2011 12:70 doi:10.1186/1471-2202-12-70