Reduced glutathione

A 10 mL overnight culture of Escherichia coli BL21 expressing pGST-SifA in LB/Ampicillin media was back diluted 1:100 into a 1 L LB/Ampicillin. The culture was grown at 37°C for 3.5 hr and then induced with 1 mM IPTG at 22°C for 3 hr. The bacteria were resuspended in GST Lysis Buffer (25 mM Tris pH 8, 150 mM NaCl, 3 mM DTT, and 1 mM PMSF) and then sonicated for 30 sec intervals 4 times. The lysate was then clarified at 14,000 rpm for 1 hr at 4 degrees Celsius. The clarified lysate was then run through glutathione sepharose beads in a column (GE Healthcare), washed with 1 X phosphate buffered solution (PBS), and then eluted using reduced glutathione (GE Healthcare).

Bacterially expressed GST-tagged CIRP and DKC1 were purified with glutathione-conjugated agarose beads, and eluted in 50 mM Tris (pH 8.0) buffer containing 50 mM reduced glutathione (GE). For EMSA, purified CIRP-GST proteins were incubated with ³²P labeled T7 capped TERC probe (Ambion) (please see Supplementary Materials for details regarding probe preparation). Binding reactions were performed at 25°C for 30 min in binding buffer (100 mM NaCl, 10 mMTris (pH7.5), 5% (w/v) glycerol, 1 mM MgCl₂, 1 U of RNasin Ribonuclease Inhibitor (Promega) and 0.3 pmol of ³²P-labeled TERC probe). The products were resolved on 5% (wt/vol) native PAGE gel (100 V, 0.5x TBE, 180 min), visualized on a Typhoon PhosphorImager (General Electric Company) and quantified using ImageQuant (Amersham Biosciences).