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. 2018 Aug 8;38(32):7032–7057. doi: 10.1523/JNEUROSCI.3542-17.2018

Figure 8.

Figure 8.

AT2R is expressed in MΦs. A, Representative agarose gel electrophoresis images of RT-PCR amplification of AT1R and AT2R genes (Agtr1a and Agtr2, respectively) from total RNA isolated from mouse MΦs and PMNs. Plasmids containing mouse Agtr1a and Agtr2 cDNAs are used as positive controls and Ly6g amplification is used for identification/validation of PMNs. Numbers on the left denote DNA molecular weight markers (in base pairs). B, Heat map showing moderate to high expression levels of RAS genes in monocytes/MΦs from RNA expression data deposited in the NCBI-GEO database. GEO mouse datasets GSE47426 (Mauer et al., 2014) and GSE57468 (An et al., 2014) and human datasets GSE10856 (Chang et al., 2008) and GSE20484 (Gleissner et al., 2010) are analyzed for RAS gene mRNA expression in monocytes/MΦs. C, Representative immunoblots depicting nonspecificity of routinely used anti-AT2R antibodies in peritoneal MΦ lysates from FVB-Agtr2-WT and FVB-Agtr2-KO mice. Anti-Iba1 and anti-mortalin antibodies are used as positive controls for MΦs and housekeeping protein, respectively. Numbers on the left denote protein molecular weight markers (in kilodaltons). D, E, Ang II (100 nm; 30 min) induces Erk1/2 phosphorylation in mouse (B6-WT) peritoneal MΦs, but not in PMNs. The AT2R inhibitor PD123319 (1 μm), but not the AT1R inhibitor losartan (1 μm), attenuates Ang II-induced Erk1/2 phosphorylation in MΦs. Ang II-induced Erk1/2 phosphorylation is absent in MΦs from FVB-Agtr2-KO mice, but intact in FVB-Agtr2-WT mice. The selective AT2R activator CGP42112A (100 nm) and TNF-α (10 nm) are used in mouse MΦs as positive controls for AT2R activation/signaling and Erk1/2 phosphorylation, respectively. LPS (10 nm) is used as a positive control for Erk1/2 phosphorylation in mouse PMNs. Mortalin (Grp75) immunoreactivity is used as loading control and the magnitude of ERK1/2 phosphorylation is quantified in E. Data are presented as individual experimental replicates, with mean ± SEM marked therein (n = 3 per group). *p < 0.05, #p < 0.05, and not significant (ns) versus indicated comparison groups, one-way ANOVA with Tukey's multiple-comparisons post hoc test.