Fig 1: Prevalence of Trx1 and TrxR1 in HIV particles.(A) Western Blot analysis to detect Trx1 and TrxR1 in the envelope of HIV-1: HIV-1(NL4.3) particles (500 ng of p24 protein) were lysed and loaded onto a 4–12% Bis-Tris PAGE gel. Trx1 and TrxR1 were detected using the antibodies 10284-RP01 and 13093-RP01, respectively. As positive controls, commercially available Trx1 and (truncated) TrxR1 were used. M: MagicMark XP Western Protein Standard. The fig shows the results of a single representative experiment.(B) Detection of Trx1 in virus particles produced in the presence of the organotellurium compounds: HIV-1 particles which were produced in the presence of 25 µM organotellurium compounds were isolated, lysed and measured for their p24 content. Subsequently, the lysates were measured for their Trx1 content via ELISA using the antibodies MT17R6 and MT13X3-biotin. The values were normalised to the p24 values. The value for the control was then set to 100% in each individual experiment and all other values were adapted against these reference values. The data are means of three independent experiments; the error bars represent the SEM. P-values are indicated if significant (*: p < 0.05; **: p < 0.01; ***: p < 0.001).
Fig 2: Inhibition of the reduction of gp120 in the presence of the test compounds.An ELISA was performed with two set-ups for each compound, the reference compound (auranofin), a vehicle control (DMSO) and DTT. In the first set-up (TrxR1 + Trx1)preincub., TrxR1 was shortly (15 min) pre-incubated with Trx1 before adding the compounds. In the second set-up (TrxR1 + compounds)preincub, TrxR1 was shortly (15 min) pre-incubated with the compounds before adding Trx1. The final reaction mixture contained 1 µM Trx1 + 100 nM TrxR1 + 240 µM NADPH + 100 µM compound or auranofin. DTT was used as a reduction control. ELISA plates were coated with recombinant human gp120. The reaction mixtures were added to the wells to allow reduction of the coated gp120, incubated for 15 min at room temperature and exposed to streptavidine-ALP and p-nitrophenyl phosphate. The value for the DTT was set to 100% in each individual experiment and all other values were normalised against this reference value. The data are means of three independent experiments; the error bars represent the SEM. P-values were calculated for each reaction in relation to its control and are indicated if significant (*: p < 0.05; **: p < 0.01; ***: p < 0.001).
Supplier Page from Sino Biological, Inc. for Human Thioredoxin / TXN / SASP Protein