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. 2014 Apr 4;9(4):e93899. doi: 10.1371/journal.pone.0093899

Figure 5. Impact of thio-redox state on matriptase activation.

Figure 5

184 A1N4 cells were homogenized in phosphate buffer pH(A) The insoluble fraction was re-suspended and incubated in phosphate buffer pH 6.0 for 20 min either on ice as a negative control (lane 1) or at room temperature as a positive control (lane 2). The insoluble fraction was also mixed with the cytosolic fraction alone (lane 3) or in the presence of various concentrations of DTNB (lanes 4, 5, and 6). Cytosolic fractions were also prepared from LNCaP and PC3 prostate cancer cells and either dialyzed or stored at 4°C for three days. The insoluble fraction prepared from 184 A1N4 cells, was incubated with the untreated, stored (S.), or dialyzed (D.) cytosolic fraction from LNCaP human prostate cells (lanes 7, 8, and 9) and PC3 prostate cancer cells (lanes 10 and 11). (B and C) The insoluble fraction of 184 A1N4 cells was incubated in phosphate buffer pH 6.0 for 20 min either on ice as a negative control (lane 1) or at room temperature as a positive control (lane 2). The insoluble fraction was also incubated with the cytosolic fraction alone (lane 3) or in the presence of increasing concentrations of oxidized glutathione GSSG (B, lanes 4, 5, and 6) or NEM (C, lanes 4, 5, and 6). Lysates were prepared from all incubation conditions and assayed by immunoblot analyses for matriptase activation.