A Fragment of Apolipoprotein E4 Leads to the Downregulation of a CXorf56 Homologue, a Novel ER-Associated Protein, and Activation of BV2 Microglial Cells

Figure 3

Confirmation of CXorf56 knockdown in BV2 microglial cells following treatment with siRNAs. (a) BV2 cells were treated for 5 hours with a scrambled siRNA sequence (Neg. siRNA Ctl, green bar) or two designed sequences (siRNA2, blue bar or siRNA3, red bar) targeted to the CXorf56 open reading frame. Following treatment, total RNA was extracted and RT qPCR was performed. In contrast to the negative siRNA control, siRNA(2) and siRNA(3) led to a 40% and 58% downregulation, respectively, of CXorf56 mRNA. denotes significant difference between scrambled negative control and siRNA(2), . # denotes significant difference between negative control and siRNA(3), . (b) Western blot analysis utilizing an anti-CXorf56 antibody (1 : 500) confirmed the decreased expression of the CXorf56 protein following treatment of BV2 cells for 24 hrs with siRNA2 (middle lane) or siRNA3 (far right lane). Beta-actin was employed as a loading control utilizing an anti-beta-actin antibody (1 : 50K), bottom panel. Data are representative of three independent (c–f) Representative confocal immunofluorescence images utilizing an anti-CXorf56 antibody in BV2 cells following treatment with siRNAs for 24 hours with nontreated cells (c), negative siRNA control (d), siRNA(2) (e), and siRNA(3) (f). A decrease in staining intensity with the anti-CXorf56 antibody was observed following treatment with siRNA2 (e) and to a lesser extent with siRNA3 (f). Note also the apparent change in morphology following treatment with extended, bipolar pseudopods in (e) and (f).

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