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Fig. 7. MLK3 enhances golgin-160 cleavage at aspartate at residue 139. HeLa cells stably expressing EGFP-tagged golgin-160 were transfected with wild-type (WT) or catalytically inactive (KR) MLK3. Cell lysates were fractionated into soluble cytosolic and membrane protein fractions, and 10% of each fraction was analysed by immunoblotting. (A) (Top) An immunoblot for GFP. The arrows indicate the position of full-length EGFP-tagged golgin-160 and EGFP/golgin-160(1-139). The presence of the caspase inhibitor Boc-D-FMK (10 µM) in wild-type MLK3 transfected cells (WT/CI) during the last 24 hours of incubation inhibited production of the golgin-160 cleavage product in the cytosolic protein fraction. Protein loading controls show the expression of p97 golgin and {alpha}-tubulin. MLK3 was found primarily in association with the membrane fraction. (B) (Top) A GFP immunoblot of the cytosolic fraction from stable cells transfected with wild-type or inactive MLK3 and compared with an EGFP-tagged golgin-160 mutant spanning residues 1-139. (Middle) Immunoblot of golgin-160 using an antibody specific for the N-terminus. (Bottom) Immunoblot for {alpha}-tubulin as a protein loading control. (C) Immunoprecipitation of EGFP-tagged proteins from soluble cytosolic fraction isolated from stable cells transfected with wild-type or inactive MLK3 using a monoclonal GFP antibody. EGFP-tagged proteins were visualized following immunoblotting with a polyclonal GFP antibody. Data represent at least three separate experiments.