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. 2016 Aug 11;291(39):20427–20439. doi: 10.1074/jbc.M116.744656

FIGURE 5.

FIGURE 5.

Search for specific ACAT1 antibodies. A, comparison of the specificity of ACAT1 antibodies from different vendors as assessed by Western blotting analysis. Retinal homogenates from Acat1−/− (negative control) and Acat1+/+ mice (3–4 months old) were run side by side on SDS-PAGE. Membranes, after transfer and blocking, were cut and probed separately with primary antibodies from Sigma, Novus, Abcam, and Santa Cruz Biotechnology. The immunoreactive signal for ACAT1 is in red, and that for GAPDH (served as a loading control) is in green. Primary antibodies from Sigma, Novus, and Santa Cruz Biotechnology showed similar immunoreactivity for ACAT1 in retinal homogenates from Acat1−/− and Acat1+/+ mice and recognized proteins with molecular masses different from that of full size ACAT1 (∼64 kDa). Only primary antibody from Abcam did not show any immunoreactivity for ACAT1 in both Acat1−/− and Acat1+/+ retinal homogenates. B, comparison of the specificity of anti-ACAT1 antibodies from Sigma, Novus, Santa Cruz Biotechnology, and Abcam as assessed by control stains with the nonimmunized (NI) serum and the secondary antibody (2o), respectively, and immunohistochemical stains of Acat1−/− retinal sections. Nuclei were stained with DAPI (in blue). Only primary antibodies from Santa Cruz Biotechnology and Abcam did not show any immunoreactivity for ACAT1. Because the primary antibody from Abcam also did not show any immunoreactivity for ACAT1 in Acat1−/− retinal homogenates, this antibody was chosen for further testing on retinal sections from different mouse genotypes. C, control stains with secondary antibody and retinal immunolocalization of ACAT1 with primary antibody from Abcam in Cyp27a1−/−Cyp46a1−/− mice (6–8 months old). Nuclei were stained with DAPI (in blue). Enlarged images of the boxed regions are also shown. Only a weak punctate signal was observed in frozen retinal sections, which was mainly localized to the OS with minor punctate immunoreactivity in the IS. Retinal paraffin sections showed a much stronger anti-ACAT1 immunoreactivity, which was localized only to the OS. Scale bars 100 μm.