Af2848

Co-transfected HEK293 cells (described above) were aliquotted and seeded at a density of 10,000 cells/100 μl/96-well for 4 h, and then treated with various concentrations of XMD8-92 for 2 h at 37 °C in a cell incubator (Thermo). After washing out cell culture medium, 110 μl/well of RIPA-cocktail buffer was added to lyse the cells overnight. On the same day, 96-well NUNC plates (PerkinElmer, AAAND-0001) were coated with 100 μl/well of MAPK7 capture antibody (R&D, AF2848) at 1 μg/ml at 4 °C overnight. Next day, plates were then washed with buffer (PBS containing 0.05 % Tween20) 3 times, and blocked with 2 % BSA-wash buffer for 1 h. Cell lysates were then added (100 μl/well) for 2 h, followed by washing 3 times. pMAPK7 (T218/Y220) detection antibody (Santa Cruz, sc-135761) was added at 5 μg/ml for 2 h, and plates then washed before incubating with secondary antibody (PerkinElmer, AD0207) for 1 h. Plates were given a final wash, enhancement solution added (PerkinElmer, 1244-104) for 30 min, before reading on the Envision reader(PerkinElmer).

For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose. Lysates were separated by SDS–PAGE on 8% Tris‐glycine gels (8% [w/v] acrylamide, 0.4 M Tris–HCl pH 8.8 0.08% [w/v] SDS, 6.2% [v/v] glycerol 0.05% [v/v] temed, and 0.02% [w/v] APS). Gels were resolved for 90 min at 35 mA and transferred to 0.2 μm nitrocellulose membrane (Hybind‐C, GE Healthcare, Amersham, UK) for 2 hr at 125 mA. Membranes were blocked in 5% (w/v) bovine serum albumin (BSA) diluted in Tris‐buffered saline (TBS) with 0.1% (v/v) Tween‐20 (TBST) and probed with primary antibodies, directed against: ERK5 (#3372), phospho ERK1/2 (#4370), GAPDH (#5174), phospho ERK5 (T218/Y220) (#3371), ERK1/2 (#4695) were purchased from New England Biolabs (Hitchin, UK). Antibodies to RAP1 (SC‐65) and unprenylated RAP1A (SC‐1482) were purchased from Santa Cruz Biotechnology. Antibodies to MEKK2 (ab33918), MEKK3 (ab40750), and MEK5 (ab45146) were purchased from Abcam (Cambridge, UK). Antibody to ZO‐1 (40‐2200) was purchased from Invitrogen (Thermo Fisher Scientific). All antibodies were diluted in 1% (w/v) BSA and incubated overnight at 4°C. Proteins were detected using rabbit‐ or goat‐specific HRP secondary antibodies (Jackson Immunoresearch Labs) and enhanced‐chemiluminescence (ECL) Western blotting detection reagent (Pierce).