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  Indian J Med Microbiol
 

Figure 2: The expression of TvAtg3 in HG and glucose-restricted cultivation. (a) The coding sequence of TvAtg3 was cloned into pTrcHis vector, which was introduced into E. coli for protein expression. The expression of recombinant protein was induced by isopropyl-β-D-thiogalactopyranoside for 1, 2, and 3 h. The His-tagged recombinant protein was separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by Western blotting using anti-His antibody. (b) The recombinant TvAtg3 protein was purified by His-bound resin column chromatography and analyzed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Lane Ct, control recombinant protein; Lane S, soluble fraction; Lane FT, flow-through fraction; Lane W, wash fraction. The numbers represent each fraction collected from elution. (c) The raised TvAtg3 polyclonal antibody (α-TvAtg3) was used to recognize the recombinant protein (rTvAtg3) for determination of the specificity. (d) The protein expression of TvAtg3 in high-glucose medium and glucose-restricted cultivation. Cell lysates collected from the high-glucose medium and glucose-restricted cultures with different incubation time were fractionated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the expression of TvAtg3 was analyzed by Western blotting analysis using anti-TvAtg3 antibody. (e) The protein expression of TvAtg3 in glucose-restricted-cultured cells treated with Wort. Trophozoites cultured under glucose-restricted (24 and 48 h) were treated with 100 μM Wort and the expression of TvAtg3 was determined compared with that of the DMSO-treated control (Ct). The expression of GAPDH was used as an internal for Western blotting. Quantitation of TvAtg3 expression was presented as a fold change to GAPDH

Figure 2: The expression of TvAtg3 in HG and glucose-restricted cultivation. (a) The coding sequence of TvAtg3 was cloned into pTrcHis vector, which was introduced into <i>E. coli</i> for protein expression. The expression of recombinant protein was induced by isopropyl-β-D-thiogalactopyranoside for 1, 2, and 3 h. The His-tagged recombinant protein was separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by Western blotting using anti-His antibody. (b) The recombinant TvAtg3 protein was purified by His-bound resin column chromatography and analyzed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Lane Ct, control recombinant protein; Lane S, soluble fraction; Lane FT, flow-through fraction; Lane W, wash fraction. The numbers represent each fraction collected from elution. (c) The raised TvAtg3 polyclonal antibody (α-TvAtg3) was used to recognize the recombinant protein (rTvAtg3) for determination of the specificity. (d) The protein expression of TvAtg3 in high-glucose medium and glucose-restricted cultivation. Cell lysates collected from the high-glucose medium and glucose-restricted cultures with different incubation time were fractionated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the expression of TvAtg3 was analyzed by Western blotting analysis using anti-TvAtg3 antibody. (e) The protein expression of TvAtg3 in glucose-restricted-cultured cells treated with Wort. Trophozoites cultured under glucose-restricted (24 and 48 h) were treated with 100 μM Wort and the expression of TvAtg3 was determined compared with that of the DMSO-treated control (Ct). The expression of GAPDH was used as an internal for Western blotting. Quantitation of TvAtg3 expression was presented as a fold change to GAPDH