Radio-immunoprecipitation assay (RIPA) buffer is a commonly used cell lysis reagent, due to its robustness in lysis and extracting proteins (abcam®). Nowadays, the original isotopic assay method is rarely performed, however the acronym for this buffer endured. The presence of three non-ionic and ionic detergents results in a vigorous lysis buffer. As RIPA buffer was utilised in the preparation of protein lysates for Western blotting, it was also initially used in the preparation of immunoprecipitation lysates (Figure 5.1).
Immunoprecipitation was conducted in order to initially precipitate, isolate and concentrate ERK5 out of the HDMEC or HeLa protein lysates with the use of an antibody previously bound to agarose beads. Subsequently, SDS-PAGE and Western blotting was utilised in an attempt to ascertain potential interacting partners of ERK5. MEK5 has previously been shown to bind ERK5, via the PB1 domain and 34 amino acid C-terminal extension of MEK5 (Nakamura et al., 2006), hence blotting for MEK5 acts as a positive control for the ERK5 IP experiments.
Furthermore, data from the previous chapter highlighted a potential role for MEK5 in VEGFR-2 internalisation and degradation in HDMEC and as a consequence, the receptor may also be in close proximity to ERK5. In contrast, no association was observed between MEK5 kinase activity and EGFR-1 in the previous chapter, thus blotting for EGFR-1 after IP could provide evidence to confirm this theory. Additionally, an antibody against AKT was also utilised in order to further elucidate the potential interaction between ERK5 and AKT in HDMEC.
It was evident that in both the HeLa and HDMEC, relatively low levels of ERK5 expression were detected in both the IP and total lysates after RIPA lysis (Figure 5.1). The high detergent content of RIPA results in a highly denaturing lysis buffer, deeming it somewhat incompatible for its use in IP. Consequently, a sucrose lysis buffer was considered for the preparation of IP lysates as it stabilises lysosymal membranes and reduces the release of proteases (Figure 5.2).
Chapter Five: Proteomic analysis of ERK5-interacting proteins in HDMEC
Figure 5.1 Immunoprecipitation of endogenous ERK5 after lysis with RIPA buffer.
HeLa and HDMEC were seeded on 10 cmdishes for 24 h, prior to overnight serum starvation. Cells were then stimulated with EGF or VEGF (50 ng/mL) for 10 or 30 min, followed by RIPA lysis. Lysates were immunoprecipitated (IP) overnight, at 4°C with an antibody against ERK5 (AF2848) that had been previously bound to Protein G Plus agarose beads. The beads were then spun down and washed, prior to the addition of 1 volume of LDS and boiling at 90°C. Samples were separated on 4- 12% NuPAGE® gels and membranes were incubated with antibodies against VEGFR-2+EGFR-1, ERK5, AKT and MEK5 for Western blotting (WB). Arrows indicate the protein band of interest amongst other non-specific bands. This result is representative of three individual experiments.
Cells lysed with the sucrose lysis buffer displayed higher levels of ERK5 in both the IP and total lysates, compared to that detected after RIPA lysis (Figure 5.2). The presence of proteins additional to AKT and MEK5 detected in the total lysates, were not considered problematic due to the enhanced detection of our proteins of interest. However, in both lysis methods, the presence of the heavy IgG-γ chains from the un-coupled ERK5 antibody (50-55 kDa) obscured the detection of AKT and MEK5 in the IP lysates (Figure 5.1 and Figure 5.2).
Chapter Five: Proteomic analysis of ERK5-interacting proteins in HDMEC
Figure 5.2 Immunoprecipitation of endogenous ERK5 after lysis with sucrose buffer.
HeLa and HDMEC were seeded on 10 cmdishes for 24 h, prior to overnight serum starvation. Cells were then stimulated with EGF or VEGF (50 ng/mL) for 10 or 30 min, followed by sucrose lysis. Lysates were immunoprecipitated (IP) overnight, at 4°C with an antibody against ERK5 (AF2848) that had been previously bound to Protein G Plus agarose beads. The beads were then spun down and washed, prior to the addition of 1 volume of LDS and boiling at 90°C. Samples were separated on 4- 12% NuPAGE® gels and membranes were incubated with antibodies against VEGFR-2+EGFR-1, ERK5, AKT and MEK5 for Western blotting (WB). Arrows indicate the protein band of interest amongst other non-specific bands. This result is representative of three individual experiments.
This could be overcome with the use of an agarose conjugated ERK5 antibody (sc-1284 AC) to prevent free antibody, and subsequently free IgG-γ chains, from concealing the 50kDa region. However, this pre-conjugated antibody proved somewhat less effective in immunoprecipitating ERK5 than the one originally used (AF2848) (Figure 5.3). Additionally, very low expressions of co-precipitating proteins were detected in endogenous levels (data not shown). Figure 5.3 illustrates the various antibodies tested for efficient IP of ERK5, as well as the Protein G Plus agarose beads control.
Chapter Five: Proteomic analysis of ERK5-interacting proteins in HDMEC
Figure 5.3 Antibodies against ERK5 utilised to ascertain immunoprecipitation efficiency.
HDMEC were seeded on 10 cmdishes for 24 h, prior to overnight serum starvation. Cells were then stimulated with VEGF (50 ng/mL) for 10 min, followed by sucrose lysis. Lysates were immunoprecipitated (IP) overnight, at 4°C with antibodies against ERK5 (#3372 NEB, sc-1284 AC SC, AF2848 R&D, sc-1284 SC and sc-1286 SC) that had been previously bound to Protein G Plus agarose beads, alongside a Protein G plus agarose beads only control. The beads were then spun down and washed, prior to the addition of 1 volume of LDS and boiling at 90°C. Samples were separated on 4- 12% NuPAGE® gels and membranes were incubated with an antibody against ERK5 (#3372 NEB).