The BCA protein assay (Thermo Fisher) was used to
determine the protein concentration of each of the cleared lysates. A 30 μg sample of each caecum or colon lysate protein was boiled for 5 min in reducing sample buffer containing DTT and resolved by SDS–PAGE, transferred to PVDF membranes and probed with the indicated antibodies. The membranes were exposed to enhanced chemifluorescence substrate (GE Healthcare, Piscataway, NJ), followed by scanning on a Typhoon Trio+ imaging system (GE Healthcare) to obtain a digital image of the probed protein. The bands were then quantified with ImageQuant software Carfilzomib (GE Healthcare). Caecum and colon snips obtained from untreated and C. difficile-infected mice were homogenized with a rotor/stator-type homogenizer while immersed in TRIzol RNA reagent (Life Technologies, Grand Island, NY). The TRIzol RNA reagent and the RNeasy Mini kit (Qiagen, Valencia, CA) were used in successive steps to isolate RNA from the caecum and colon samples, each according to its manufacturer’s instructions. An Agilent Bioanalyser (Agilent Technologies, Palo Alto, CA) and a Nanodrop instrument (Thermo Fisher) were used to determine Pembrolizumab order the quality and concentration of each RNA isolate, respectively.
Complementary DNA (cDNA) was generated from each RNA sample using the RT2 First Strand kit (Qiagen). Expression levels of the genes under study were determined by using two different sets of mouse RT2 Profiler PCR cards (Qiagen), each custom-made to contain eight replicate sets of
48 primer pairs (Table 1). Each well of the replicate sets was loaded with 5 ng of cDNA reaction product. Each card was run on a LightCycler 480 real-time PCR system (Roche). The relative RNA expression levels were inferred from the Ct values. Xbp1 splicing was assessed as previously described. Briefly, the Superscript III RT-PCR kit (Life Technologies) was used to amplify both unspliced and spliced Xbp1 in RNA samples obtained at the end of the experimental period. The primers used in the assay flanked the Xbp1 intron and had the following sequences: upstream: ttgtggttgagaaccagg; downstream: tccatgggaagatgttctgg. Quantitative RT-PCR, including methods for verifying primer efficiency and specificity, were performed as previously described. The Ct value for each gene Org 27569 of each sample was normalized against the geometric mean of the Gapdh and Hprt for that sample. For the following assays, differences between untreated and C. difficile-infected mice were evaluated for significance by using paired t-tests at P ≤ 0.05: diversity of the bacterial community examined by pyrosequencing; cell numbers obtained by analysing the flow cytometric data; mRNA expression for the UPR genes Gadd34 and Wars obtained by single gene quantitative RT-PCR; and protein expression or phosphorylation assessed by immunoblotting.