Brucella Induces an Unfolded Protein Response via TcpB That Supports Intracellular Replication in Macrophages
Figure 2
Brucella infection activates the UPR in macrophages in vitro.
RAW 264.7 macrophages were uninfected (NI) or infected with 100 MOI B. melitensis (B. mel). A) After 24 h, cells were resuspended in TRIzol for RNA processing. Relative expression of reverse transcribed cDNA was determined by quantitative PCR (qPCR) with normalization to 18S rRNA or hprt. Bars are combined mean fold inductions for 4–5 independent experiments (NI = 1) ± sem. *P<0.05, **p<0.003. B) Cells were infected with 100 MOI for 24 h and processed for RNA. XBP1 spliced and unspliced mRNA species were resolved by high-density agarose gel or detected by qPCR. %Splicing = spliced/total×100. Bars represent combined mean ± sem from 2–4 independent experiments. Representative gel is shown below. C) 16 h post infection, RAW cells were lysed and lysates resolved by SDS PAGE. CHOP or β-actin proteins were detected by immunoblot. Results are representative of 3 experiments.