Figure 2.

Taqman real-time RT-PCR analysis of ER stress genes in mock-, Fr57E-, and FrCasE-infected microglia. Panel A shows a gene expression comparison of FrCasE-, Fr57E- and mock-infected cultured microglia (Mg) for a subset of genes previously shown to be differentially expressed in the brains of FrCasE-infected mice compared to mock- and F43/Fr57E- infected controls [29]. Panel B shows a Western Blot for CHOP/GADD153 protein expression levels in equivalent samples from cultured microglia with mock, Fr57E and FrCasE virus infection, with and without 1 μM thapsigargin treatment for 24 hours in culture. For comparison purposes equivalent samples from mixed glial cultures (1°CNS) were assayed in parallel (right). No differences in CHOP expression were noted between mock-, Fr57E-, and FrCasE infected microglia or 1°CNS cultures, with or without thapsigargin treatment when evaluated in triplicate (not shown). Panel C shows Taqman real-time RT-PCR analysis on enriched microglial fractions taken from mock-, F43-, or FrCasE-infected brains of IRW mice 14 dpi. RT-PCR was performed on three biological replicates for each gene, and each replicate was amplified in three separate reactions and normalized to either GAPDH or ribosomal protein S29. Viral RNA was also assessed in these samples with primers and probes specific for the Friend FB-29 viral background common to FrCasE and Fr57E. F4/80 message levels were used to assess microglial enrichment, which was greater than 50 fold. Black bars = FrCasE-infected; Gray bars = Fr57E infected; white = mock infected.