Journal: The FEBS journal

Article Title: GTP binding and hydrolysis kinetics of human septin 2.

doi: 10.1111/j.1742-4658.2006.05333.x

Figure Lengend Snippet: Fig. 2. SEPT2 is phosphorylated at S218 by casein kinase II. (A) In-gel kinase assay reveals that Sept)2 is an efficient substrate for kinases that have a mobility between 40 and 50 kDa. Gels were polymerized with GST (left lanes) or GST-SEPT2 (right lanes) and duplicate samples of HeLa cell lysates were electrophoresed. The gels were renatured in the presence of [32P]-ATP as described in the methods and then autoradiographed. (B) In-gel kinase assays show that commercial casein kinase II (left panel, right lane) gave bands comparable to the cell ly- sate (left panel, left lane) and both bands were inhibited by heparin (right panel). (C) Increasing concentrations of TBB reduced SEPT2 phos- phorylation. Cells were transiently transfected with myc-SEPT2, then immunoprecipitated, labeled with [32P]-orthophosphate, and then lysed and lysates immunoprecipitated with anti-myc IgG. Increasing amounts of TBB caused a reduction in the labeling of myc-SEPT2. (D) Cells were either mock transfected or transfected with myc-SEPT2, labeled with [32P]-orthophosphate, and then immunoprecipitated with anti- myc as above. Autoradiography reveals that the wild-type protein is significantly labeled but SEPT2S218A is not (top panel). A western blot of lysates was probed with anti-SEPT2 to reveal that both constructs were equivalently expressed (lower panel, upper band), and at levels not greatly above endogenous SEPT2 levels (lower panel, lower band).

Article Snippet: Cells were then grown in the presence of 0.75 mCi [32P]-orthophosphate for 5 h at 37 C. Cells were lysed with lysis buffer (40 mm Tris, pH 7.5, 20% glycerol, 0.2 m NaCl, 2 mm EDTA, 0.5% Triton X-100, 1 lm okadaic acid sodium salt, 50 mm NaF, 0.4 mm orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 1 lgÆmL)1 of each of the protease inhibitors leupeptin and pepstatin A. SEPT2 was immunoprecipitated with mouse antic-myc monoclonal antibody IgG1 (9E10, Santa Cruz biotechnology, Santa Cruz, CA, USA).

Techniques: Kinase Assay, Transfection, Immunoprecipitation, Labeling, Autoradiography, Western Blot, Construct