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Kinoshita and colleagues reported that phosphorylated proteins could be visualised with the use of an alkoxide-bridged dinuclear metal (Mn2+_{) complex as a novel phosphate-} binding tag molecule (Phos-tag™_{) (Kinoshita et al., 2006). This Phos-tag}™ _{reagent has a} vacancy on the two metal ions, which provides suitable access for capturing phosphomonoester dianions bound to Ser, Thr and Tyr residues. Thus, phosphorylated proteins bound to Phos-tag™ _{migrate slower through a polyacrylamide gel than their} corresponding non-phosphorylated proteins, resulting in the visualisation of multiple bands during Western blotting (Figure 3.3) (Kinoshita et al., 2006).

Figure 3.3 Mode of action of Phos-tag acrylamide

Image from: http://www.wako-chem.co.jp/english/labchem/product/life/Phos-tag/Acrylamide.htm

The recommended concentrations for the Phos-tag™ _{reagent were 10% (w/v) acrylamide,} 100 µM Phos-tag and 140 µM MnCl2. However, with the equipment available – XCell SureLock™ _{and XCell II}™ _{Blot system – the substantial heat produced during electrophoresis} rendered the gel unusable. Hence it was necessary to optimise the conditions for protein electrophoresis and transfer. During optimisation of these conditions, it became apparent that a lower total concentration of acrylamide present (%T; see Table 3.1) in the gel would enhance the electrophoretic mobility of both the non-phosphorylated and phosphorylated protein bands, whilst enabling the reduction in the amount of Phos-tag™

Chapter Three: Characterisation of ERK5 activation

reagent used. Furthermore, it proved valuable to increase the ratio of Mn2+ _{ion to Phos-} tag™ _{to an excess of 3:1, thus ensuring all reagent molecules were primed to capture} phosphorylated proteins.

The Phos-tag™ _{optimisation conditions that were unsuccessful in distinctly separating the} phosphorylation events of ERK5, are indicated in normal type font in Table 3.1 (data not shown).

Table 3.1 Optimisation Phos-tag™ _{SDS-PAGE conditions} %T (w/v)

Acrylamide gel

Final Phos-tag™

concn _(µM) Final MnCl2 _concn _(µM)

Electrophoresis system, amperage &

time

Western blot system, amperage &

time

10% 100 200 XCell SureLock_{30mA, 2h} ™; XCell II_{125mA, 2h} ™ Blot;

8% 100 200 XCell SureLock_{10mA, 6h} ™; XCell II_{125mA, 2h} ™ Blot;

8% 70 210 XCell SureLock_{10mA, 6h} ™; XCell II_{125mA, 2h} ™ Blot;

8% 70 210 XCell SureLock_{8mA, 22h} ™; XCell II_{125mA, 2h} ™ Blot;

7% 70 210 XCell SureLock_{8mA, 22h} ™; XCell II_{125mA, 2h} ™ Blot;

7% 70 210 Mini-PROTEAN_{8mA, 22h} ®; Mini Trans-Blot_{125mA, 2h} ®;

7% 70 210 Mini-PROTEAN_{8mA, 22h} ®; XCell II_{125mA, 2h} ™ Blot;

After these initial experiments, it was evident that the XCell II™ _{Blot Module enabled} clearer transfer of proteins onto the nitrocellulose membrane than the Mini Trans-Blot®_. Thus, the XCell II™ _{Blot was utilised in an experiment that compared the quality of ERK5} phosphorylation band separation using the XCell SureLock™ _{and Mini-PROTEAN}® electrophoresis systems, and the optimised conditions indicated in bold type font in Table 3.1 (Figure 3.4).

EGF-stimulated HeLa and VEGF-stimulated HDMEC protein lysates were separated for 22 h at 8 mA on a 7% (w/v) acrylamide, 70 µM Phos-tag™_{, 210 µM MnCl2 gel either using the} XCell SureLock electrophoresis system or the Mini-PROTEAN system. However, both gels were transferred using the XCell II Blot Module at 125 mA for 2h prior to Western blotting with an antibody against ERK5 (Figure 3.4).

Chapter Three: Characterisation of ERK5 activation

Using the XCell SureLock system, it was apparent that the protein lysates did not migrate through their lanes in a stable manner, as the bands were not horizontally aligned (Figure 3.4a). This was attributed to inefficient heat conduction from the plastic cassettes to the surrounding buffer, produced during electrophoresis. Nevertheless, in both cell types and all conditions, a higher migrating band (*) that did not fully resolve from the ERK5 band was observed and was termed “p-ERK5” in Figure 3.4a.

Figure 3.4 Optimisation of Phos-tag™ _{gel SDS-PAGE to detect ERK5 activation.}

HeLa cells and HDMEC were seeded on 6-well plates for 24 h, prior to overnight serum starvation. HeLa were stimulated with EGF (50 ng/mL) and HDMEC with VEGF (50 ng/mL), for 10 or 30 min, followed by RIPA lysis. Lysates were separated for 22 h at 8 mA on a 7% (w/v) acrylamide, 70 µM Phos-tag™_{, 210 µM MnCl2 gel using either (A) an Invitrogen XCell SureLock}™ _{Mini-Cell electrophoresis} system or, (B) a Bio-Rad Mini-PROTEAN® _{Tetra Cell electrophoresis system. Both gels were} transferred for 2 h at 125 mA, using an Invitrogen XCell II™ _{Blot Module prior to Western blotting} (WB) with an antibody against ERK5. Densitometric analysis of protein phosphorylation or protein expression relative to the basal control condition of each cell type, is displayed beneath each blot. The basal control condition for each cell type and each migrated form of ERK5 was set arbitrarily as 1.0.

The HeLa cells exhibited an additional band migrating much slower than the p-ERK5 band suggesting this represented a hyper-phosphorylated ERK5 and was designated the “hyper p-ERK5” band (Figure 3.4a). This band was present in unstimulated HeLa, but significantly increased in intensity at 10 min EGF stimulation and then somewhat decreased again at 30 min stimulation, yet still remained more intense than the unstimulated condition. Importantly, this hyper p-ERK5 band was not detected in unstimulated or VEGF-stimulated HDMEC (Figure 3.4a).

Chapter Three: Characterisation of ERK5 activation

Protein separation using the Mini-PROTEAN® _{system appeared to result in greater stability} of migration, as all detected bands were horizontally aligned. Furthermore, all observed bands were distinct from one another, making it possible to quantify their intensity relative to the respective basal condition migration band for each cell type (Figure 3.4b).

EGF stimulation of HeLa for 10 min resulted in a faint band that migrated slower than that of ERK5, suggestive of a phosphorylation event and was termed “p-ERK5” (Figure 3.4b), however at 30 min post-stimulation, this band disappeared. This p-ERK5 band was also present in VEGF-stimulated HDMEC at 10 min, remaining sustained for 30 min.

A clear hyper p-ERK5 band was observed in all conditions of HeLa cells whilst using the Mini-PROTEAN® _{system (Figure 3.4b), the intensity of which, followed that observed in} Figure 3.4a. Once again however, this band was not detected in either the unstimulated or VEGF-stimulated HDMEC conditions (Figure 3.4b). Overall these data suggested a potential difference in the agonist-stimulated phosphorylation of ERK5 in HeLa and HDMEC.