Storm phosphorimager

Total genomic DNA was digested with SacII (New England BioLabs) for strains containing pBR322-derived plasmids or NheI (New England BioLabs) for pCB104-derived plasmids. In both cases, plasmids lack restriction sites for these enzymes. Samples were then extracted with one volume of chloroform before equal cell equivalents were electrophoresed through 1.0% agarose gel in 1× TBE (Tris-borate-EDTA, pH 8.0) at 1 V/cm. DNA in the gels was transferred to a Hybond N+ nylon membrane, and the plasmid DNA was detected by probing with either ³²P-labeled pBR322 or pCL01 plasmid DNA prepared by random-primer labeling (Agilent Technologies) using α³²P-labeled-dCTP (3000 Ci/mmol; PerkinElmer) and visualized using a STORM PhosphorImager with its associated ImageQuant analysis software (Amersham Biosciences).

Arabidopsis thaliana Col-0 suspension cells were cultivated as described by Krinke et al. (2009 (link)). Experiments were performed on 5-day-old cultures. Suspension cells were labeled with ³³P_i according to the procedure previously described by Krinke et al. (2007 (link)). Total lipids were extracted and separated by thin layer chromatography (TLC). Structural phospholipids and PA were separated in the acid solvent system composed of chloroform-acetone-acetic acid-methanol-water (10:4:2:2:1, v/v/v/v/v) (Lepage, 1967 (link)). Phosphoinositides were separated in the alkaline solvent system composed of chloroform-methanol-5% (w/v) ammonia solution (9:7:2, v/v/v), where the TLC plates were soaked in potassium oxalate solution before heat activation (Munnik et al., 1994 (link)). Radiolabeled spots were quantified by autoradiography using a Storm Phosphorimager (Amersham Biosciences). Separated phospholipids were identified by co-migration with authentic non-labeled standards visualized by primuline staining (under UV light) or by phosphate staining.

This was performed as described previously^{18 (link)}. Full-length or truncated versions of TBX5 proteins were generated as MBP-fusions in bacteria following IPTG induction. These fusion proteins were partially purified from bacterial lysates on amylose resin. Their integrity was checked on PAGE by Coomassie blue staining of the gel. MBP-TBX5 fusion proteins were immobilized on amylose resin and GST-MEF2C was immobilized on glutathione sepharose beads for interaction studies. Equivalent amounts (2–5 μg) of immobilized MBP or GST fusion proteins were incubated with ³⁵S-labelled full-length MEF2C or TBX5 (wild type or mutant) in binding buffer (50 mM Tris pH 7.4, 100 mM NaCl, 0.05% NP40, 1 mM DTT, 10% glycerol, 0.5 mM PMSF, 1μg/ml aprotinin, 1μg/ml pepstatin, 1μg/ml leupeptin, 0.05% BSA) for 2 hours at 4 °C. Beads were washed four times with binding buffer without BSA before the bound proteins were released from the beads by boiling with SDS sample buffer. Eluted proteins were subsequently resolved on SDS-PAGE followed by scanning on a Storm PhosphorImager (Amersham Bioscience), and band intensity was quantified using Image Quant software (Molecular Dynamics).