1. Supplementary figures
Supplementary Figure 1. Effect of buffer exchange on purified PTEN phosphatase activity. The activity of the stock protein in elution buffer (50 mM Tris pH 7.4, 20 mM reduced L-glutathione, 250 mM NaCl, 2 mM DTT in 50% glycerol) was compared to the activity of the untreated protein following buffer exchange in 20 mM Tris pH 7.4, 0.1 mM EDTA, 100 mM NaCl, in order to assess whether accidental activity loss had taken place before the oxidative treatment. The data are presented as mean (SD) of three independent experiments. No significant difference was found in the specific activity of the protein before and after buffer exchange (two-tailed unpaired Student’s t test, p = ). The calculated specific activity values are: ± nmol OMF/min/mg protein for the stock PTEN-GST and ± nmol OMF/min-1mg-1 for the buffer-exchanged PTEN-GST.

2. Lane 1
Lane 2
Lane 3
Lane 4
Lane 5
Lane 6
Supplementary Figure 2. Representative Coomassie-stained gel showing isolation of putative PTEN-GST binding proteins by GSH-affinity enrichment following 1 mM H2O2 oxidation. Lane 1: glutathione sepharose beads incubated with HCT116 cell extract; Lane 2: glutathione sepharose beads loaded with GST incubated with HCT116 cell extract; Lane 3: glutathione sepharose beads loaded with native PTEN-GST incubated with HCT116 cell extract; Lane 4: glutathione sepharose beads loaded with H2O2 oxidized PTEN-GST incubated with HCT116 cell extract ; Lane 5: no-bead control with native PTEN-GST; Lane 6: no-bead control with H2O2 oxidised PTEN-GST.

3. A B C D
Supplementary Figure 3. Uncropped scans of the Western Blots presented in Figure 4A (A), 4B (B), 4C (C) and 4D (D). The dashed red boxes mark the borders of cropped area used for the main figure. The molecular weight markers are shown on the left. The detected protein and any oxidizing and/or reducing treatment performed on the bait PTEN-GST are indicated on the right.

4. PTEN-GST loading controls A) B) C)
Red Ox Rec
Red Ox Rec MW
130
100 70 50
Anxa2 PTEN-GST ~112kDa
Prdx1 PTEN-GST ~96kDa D) E) MW
Red Ox Rec
MW Red Ox Rec
100 70 F) 55 35 70 25 G) 70 15
Trx PTEN-GST ~86kDa
PTEN-GST loading controls
Supplementary Figure 4. Non-reducing gels blotted for target proteins to identify if association is due to disulfide crosslinking. In all cases MW is molecular weight markers, lane 1 is reduced PTEN-GST (Red), lane 2 is H2O2 oxidised PTEN-GST (Ox) and lane 3 is H2O2 oxidised PTEN-GST with activity recovered by DTT incubation (Rec). Dashed boxes indicate region on gel corresponding to expected MW of disulfide crosslinked PTEN-GST and target protein. A) MW markers; B) blot for Annexin A2, with expected MW of crosslinked proteins approximately 112kDa; C) blot for peroxyredoxin 1, with expected MW of crosslinked proteins approximately 96kDa; D) MW markers; E) blot for thoredoxin, with expected MW of crosslinked proteins approximately 86kDa; F) PTEN control blot for panels B) and C); G) PTEN control blot for panel E).