Cerni FA , Pucca MB , Peigneur S , Cremonez CM , Bordon KC , Tytgat J , Arantes EC .

	Figure 1. Elution profile of Ts venom on CM-cellulose-52 column. Absorbance was monitored at 280 nm. (A) Classical method of fractionation. Tityus serrulatus venom (200 mg) was dispersed in 2 mL of 0.05 M NH4HCO3 and the supernatant was fractionated on a column 2.5 cm × 63.0 cm, equilibrated with 0.05 M ammonium bicarbonate, pH 7.8 (Buffer A), at 4 °C. Flow: 0.3 mL/min. The dotted line represents the beginning of the convex concentration gradient of 0.01–1 M of ammonium bicarbonate (Buffer B). (B) Improved method of fractionation using a FPLC Äkta Purifier UPC-10 system. Tityus serrulatus venom (50 mg) was dispersed in 2 mL of 0.05 M NH4HCO3 and the supernatant was fractionated on a column 1.6 cm × 100.0 cm, equilibrated with 0.05 M ammonium bicarbonate, pH 7.8 (Buffer A), at 25 °C. Flow: 0.5 mL/min. The dotted line represents the beginning of the linear concentration gradient from 0% to 100% of 0.6 M of ammonium bicarbonate (Buffer B). Absorbance was monitored at 280 nm.
	Figure 2. Reversed-phase FPLC of fractions X and XIIA resulting from the improved Ts venom fractionation procedure. The fractions were purified on a C18 column (4.6 mm × 250 mm, 5 µm particles) equilibrated with 0.1% (v/v) of trifluoroacetic acid (TFA). Adsorbed proteins were eluted using a concentration gradient from 0% to 100% of solution B (80% acetonitrile in 0.1% TFA), represented by the dotted line. Flow: 0.8 mL/min. Absorbance was monitored at 214 nm, at 25 °C. (A) Fraction X; (B) Fraction XIIA.
	Figure 3. Blocking effect of Ts6 on 14 cloned potassium channels. Representative traces in the absence or presence (*) of 1 µM Ts6 are shown. Voltage protocol is detailed in Section 4.2 of Material and Methods.
	Figure 4. Blocking effect of toxins Ts6 and Ts7 on different types of potassium channels. (A) Ts6 blocking effect on 14 cloned potassium channels (n ≥ 3); (B) Ts7 blocking effect on 12 cloned potassium channels (n ≥ 3).
	Figure 5. Functional features of Ts6 on Kv1.2 and Kv1.3 potassium channels. (A) Dose-response curve of Ts6 on Kv1.2 channels using Hill equation (for each tested concentration, n ≥ 3). (B) Dose-response curve of Ts6 on Kv1.3 channels using Hill equation (for each tested concentration, n ≥ 3). (C) Current/Voltage relationship on Kv1.2 in the absence (square) or in the presence of 1 µM Ts6 toxin (circle) (n ≥ 3). (D) Current/Voltage relationship on Kv1.3 in the absence (square) or in the presence of 1 µM Ts6 toxin (circle) (n ≥ 3).
	Figure 6. Blocking effect of Ts7 on 12 cloned potassium channels. Representative traces in the absence or presence (*) of 1 µM Ts7 are shown. Voltage protocol is detailed in Section 4.2 of Material and Methods.
	Figure 7. Multiple sequence alignment and Kv block effect of α-KTx toxins including Ts6 and Ts7. The aligment and percentage of identity Id (%) of 11 primary structures were created by ClustalW2. The figure was generated by ESPript and adapted in CorelDrawn13. UniProt accession numbers are followed by the toxins names. The highly conserved residues are in red. Cysteine residues are highlighted in pink. The amino acid residues in blue indicate low consensus and those not conserved are in black. The functional dyads are demarcated (*).

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