Western Blotting

25 µg total protein from the cytosol fraction derived from each SKOV3 cell sample

was mixed with the sample buffer as described in detail in the Materials and

Methods in Chapter 2. For detecting AK2 and calreticulin, blotted membranes were

incubated with anti-AK2 or anti-calreticulin polyclonal antibodies at one in 5,000

and 10,000 dilution, respectively. To detect DP8 and DP9 proteins, anti-DP8 and

anti-DP9 polyclonal antibodies were diluted in Tris buffer solution at ratio 1: 5,000.

As a loading control, anti-human ß-actin was diluted 1: 10,000 in Tris buffer

solution. Anti-rabbit secondary antibody coupled with HRP was used to detect

antigen-antibody binding complexes for AK2, DP8 and DP9. Calreticulin primary

antibody binding complexes were detected using an anti-goat secondary antibody

with HRP. Anti-mouse secondary antibody linked to HRP was used to detect the

interaction between ß-actin and its primary antibody (Section 2.5.7.4). The

ChemiDocTM imaging system was used to visualise band images with an exposure

time of 20 sec for calreticulin and ß-actin. Other immunoblotted membranes were

exposed for 900 sec.

DPP4 enzyme activity

10µl of cell lysate, cytosol or mitochondrial fractions from cell lines were loaded

into a 96 well plate. All experimental samples were tested in triplicate. Samples

were diluted with 40µl of Tris-buffer solution which consisted of 50 mM Tris-HCl

pH 7.4 and 150 mM NaCl. 50 µl of 1mM H-Ala-Pro-pNA substrate also dissolved

in Tris-buffer pH 7.4 was added into each well. The temperature of the Fluorostar

spectrophotometer was set at 37ºC and the machine was configured to take
continual readings every 5 min for 60 min. Every 5 min, the microplate was shaken

at 500 rpm for 30 sec and the absorbance values were directly measured with 405

nm and 600 nm to reduce background. The specific enzyme activity was determined

using the Beer-Lambert formula A = εCl, where A = absorbance, ε = µ molar

extinction coefficient (9.45 litres.µmol-1.cm-1 for pNA at 405 nm), C =

concentration (µmol.litre-1) and l = length of light path (2.94 cm for Sarstedt 96 well

plate). Before calculating enzyme activity, the absorbance values of samples were

normalized with the values taken for a Tris-buffer control. Δ absorbance was

calculated by subtracting absorbance values at two different time points during the

exponential phase of enzyme activity. The Δ absorbance per minute was then

determined by