Optimisation of Endogenous Co-immunoprecipitations Methods Endogenous immunoprecipitations required significant optimisation in order to

obtain robust evidence of an in vivo interaction. Initially, immunoprecipitations were performed with antibodies against LIMD1, PHD2 and VHL. Dependent upon the epitope binding region, binding of an antibody to its target protein can disrupt endogenous protein-protein binding, and so the three different antibodies were used to avoid this issue.

As the majority of the work regarding LIMD1 and its role in hypoxic regulation had been performed in U2OS cells, these cells were again used for the initial immunoprecipitations. To obtain complete cell lysis, cells were lysed in RIPA buffer, which contains the denaturing agent SDS, the detergent NP40 and a physiological concentration of NaCl (150mM), making it a stringent buffer. Initial immunoprecipitations were performed in RIPA, followed by 4 washes of the IP- bead matrix, again with RIPA. These results indicated that the anti-LIMD1 and VHL antibodies were able to successfully immunoprecipitate their target protein (Figure 5.8A and B).

PHD2 is 48kDa in size, and due to cross reactivity of the secondary antibody with the immunoprecipitating PHD2 IgG heavy chain (molecular weight 50kDa) it was difficult to distinguish between PHD2 and the IgG heavy chain via Western blot (Figure 5.8C). Therefore until optimisation of the immunoprecipitations had been fully carried out, immunoprecipitation with this antibody was omitted.

Figure 5.8: -LIMD1 immunoprecipitate th

15cm plate of U2OS c phosphatase inhibitors matrix (Santa Cruz). An

of bound proteins with 5 x SDS buffer and analysis by Western blot. (A)

IMD1, VHL and PHD2 antibodies are able e their target proteins. For each immunoprecip

OS cells were lysed in RIPA buffer supplemented ors and incubated overnight with antibody pre-co ). Antibody-IP matrices were washed 4 times in RI of bound proteins with 5 x SDS buffer and analysis by Western blot. (A)

ble to successfully

recipitation, a confluent ted with protease and conjugated to the IP RIPA, prior to elution of bound proteins with 5 x SDS buffer and analysis by Western blot. (A) -LIMD1 and (B)

For the first experiments, the standard lab protocol was to carry out 4 x 5min washes of the IP matrix using RIPA buffer following incubation of the antibody and lysates. However, following this the co-immunoprecipitated proteins were only detectable as faint bands (Figure 5.9A). Furthermore, with the LIMD1 antibody, the amount of precipitated and co-immunoprecipitated protein was substantially less than with the VHL antibody (Figure 5.9A). Therefore, for optimisation purposes only immunoprecipitation with the VHL antibody was pursued.

Non–specific binding to the isotype control antibody was the major problem incurred with the immunoprecipitation experiments. To optimise this, the length of time the antibody was incubated with the cellular lysates was reduced from overnight (16-20 hours) to 4 hours, which significantly reduced the non specific binding (Figure 5.9B).

To further eliminate non-specific binding, whilst maintaining specific co- immunoprecipitation, the buffer for washing of the IP-bead matrix, along with the number of times the IP matrix was washed following incubation was optimised by carrying out a titration of between 2 and 6 washes. The level of co- immunoprecipited protein was high at 2 washes and systematically decreased as the number of washes increased. However, with fewer washes the level of non- specific binding to the isotype control increased. The optimal point for these two parameters was 3 washes. Furthermore, instead of using pure RIPA for the washes, a wash buffer of 50/50 PBS/RIPA was used to reduce the stringency of the washes; this helped eliminate binding of proteins to the isotype control whilst concurrently being the correct stringency so that the degree of co- immunoprecipitated proteins were maintained with the specific -VHL IP complex.

Figure 5.9: Optimisation of endogenous immunoprecipitations. (A) Following initial

overnight immunoprecipitation with anti-LIMD1, VHL and isotype control (1, -GFP; 2, - cortactin) antibodies where the IP matrix was washed 6 times, only low levels of co-

50- 75- 100- 150- kDa IgG IP: VHL Blot: LIMD1 4hr incubation 16hr incubation 50- 75- 100- 150- kDa IgG

U2OS HeLa HEK 293T

IP: VHL Blot: LIMD1 LIMD1 37- 50- 75- IP: VHL/LIMD1 Blot: PHD2 kDa 30min exposure

A

B

C

For immunoprecipitations, U2OS cells from a confluent 15cm plate were used. Contact inhibition in this cell line meant that the cell density achievable in a confluent 15cm plate of U2OS was less than that of other cell lines. To establish if cell number (i.e. amount of protein) was limiting the immunoprecipitations, 2 other well established cell lines within the lab, HeLa and HEK293T, were used in

the immunoprecipitations as in a 15cm plate format, a greater cell

number/density could be achieved compared to U2OS. The higher cell number was reflected by greater protein input levels (as detected by Western blotting

1% of the total cell lysate), and subsequently greater levels of co-

immunoprecipitated protein (Figure 5.9C).

The final step of optimisation was to increase the amount of antibody used in the immunoprecipitation from 4µg to 5µg. So far the other possibly limiting steps (antibody, number of washes, wash buffer and cell number) had been optimised. Increasing the amount of immunoprecipitating antibody made the significant difference in these experiments; the amount of co-immunoprecipitated protein

was substantially increased. This implied that the lower amount of

immunoprecipitating antibody in the initial experiments was the limiting factor and not sufficient to precipitate all of the available endogenous complexes. 5.4.4 Endogenous VHL, LIMD1 and PHD2 Co-Immunoprecipitate In

Vivo.

Immunoprecipitation of endogenous VHL from HEK 293T cells using 5µg of anti- VHL antibody, resulted in co-immunoprecipitation of endogenous LIMD1 and PHD2, demonstrating their association in an endogenous in vivo complex (Figure 5.10). In addition to having the isotype antibody as a control, two proteins that are part of the VCB complex and bind VHL, elongin B and cullin 2, also co- immunoprecipitated, further corroborating the specificity and integrity of the endogenous complex in vivo.

When VHL was immunoprecipitated, there are five bands of different molecular weights between ~19 and 35kDa observed. However, in the input lane, only two molecular weights are observed, which correspond to the characterised p19 and p25 isoforms. The other bands are of unknown origin; multiple VHL banding has been previously identified (Liu et al., 2011), but the identity of the modification was unknown. Similarly, following co-immunoprecipitation of LIMD1, as well as a molecular weight band of 75kDa that corresponds to what is observed in the input, three additional higher molecular weight bands are also observed that correspond to a modified LIMD1. However, as both LIMD1 and VHL are part of a

concentrated ubiquitin ligase complex, and VHL itself becomes ubiquitylated (Liu et al., 2011), it could be speculated that these extra forms could represent ubiquitylated VHL or LIMD1.

Figure 5.10: Co-immunoprecipitation of LIMD1, PHD2, elongin B and cullin 2 when VHL is immunoprecipitated. A confluent 15cm plate of HEK 293T cells were

HEK 293T