HEK293 were transfected with a vector encoding HA-SIK2 and lysed 48 h post-transfection. Cell lysates (200 μg protein extract) were incubated with increasing amounts of the mouse monoclonal anti-HA antibody (12ca5) for 90 min at 4°C and then immune complexes were recovered by adding 10 μl of protein G- sepharose and incubating sample for a further 30 mins at 4°C. HA-SIK2 was also immunoprecipitated by incubating whole cell extracts with 5 μl of anti-HA coupled beads from Sigma. The immunoprecipitates were washed with lysis buffer and proteins bound to the column were eluted with 1% SDS, seperated by SDS-PAGE (8% gel) and immunoblotted using the rat monoclonal antibody (3F10) from Roche.