pylori 84–183 rpsL gene in pCR II-TOPO This study pKR020 cat cassette in pKR002 This study pKR021 rpsL HP /cat construct in pKR002 This study To confirm the proteomics-implicated temperature regulation of Cj0596, a western blot was performed on C. jejuni cells grown at 37°C or 42°C using anti-Cj0596 antibody (1:1,000) as the primary antibody and HRP conjugated goat anti-rabbit IgG (1:50,000) as the secondary antibody. A control western
blot against Cj0355 (expression of which is unaffected by growth temperature (Fields and Thompson, unpublished results; ) was performed using anti-Cj0355 antibody AZD8931 (1:1,000) as the primary antibody and HRP conjugated www.selleckchem.com/products/gw3965.html goat anti-rabbit IgG (1:50,000) as the secondary antibody. The blots were developed using a DAB Substrate Kit (BD Biosciences). Densitometry measurements were conducted using ImageJ software . Localization of the Cj0596 Protein To determine the cellular location of Cj0596, C. jejuni cells grown
at 37°C were separated into cytoplasmic, periplasmic, and inner Barasertib membrane fractions , and outer membrane fractions as described . Western blots were performed on C. jejuni cell fractions using anti-Cj0596 antibody (1:1,000) as the primary antibody, along with control blots using anti-Cj0355 (cytoplasmic control),
anti-CetA (inner membrane control), and anti-MOMP (outer membrane control) polyclonal sera (all at 1:1,000) as the primary antibodies. HRP conjugated goat anti-rabbit IgG (1:50,000) was used as the secondary antibody for all blots, which were then developed using a DAB Substrate Kit (BD Biosciences). PPIase Assay The PPIase activity of Cj0596 was determined in a coupled Morin Hydrate assay, which measures the ability of Cj0596 to convert the cis isomer of the oligopeptide substrate N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide into the trans form which is cleavable by α-chymotrypsin [40–42]. Chymotrypsin (0.63 μg/ml) and varying concentrations of Cj0596 were combined in 50 mM Tris-HCl pH 7.8 and incubated at 4°C. The substrate (93.8 μg/ml) was added and the reaction was monitored at 10°C by the increase in absorbance at 390 nm (corresponding to the release of p-nitroanilide). The kobs value for each PPIase concentration was found by plotting Ln [A390(∞)-A390(t)] vs. time (sec) and determining the slope. The catalytic efficiency (k cat/K m) of the PPIase activity was obtained by plotting kobs vs. [PPIase] and determining the slope.