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. 2012 Oct 9;7(10):e46934. doi: 10.1371/journal.pone.0046934

Figure 8. Down-regulationof Pcsk1n rescued the effect of Pax6 knock-down on PC1/3 and proinsulin processing.

Figure 8

A, MIN6 cells were transfected with Pax6, Pax6 and Pcsk1n or scrambled siRNA in triplicate, and 48 hours later protein was analyzed by western blot. B, ratio of densitometry scanning of 87 kDa and 66 kDa PC1/3 bands is shown (n = 3). C, enzyme activity of PC1/3 was examined in Pax6 RNAi or Pax6 and Pcsk1n double RNAi treated MIN6 cells with equivalent protein. Scrambled siRNA was used as negative control (n = 3). Black circles, values for transfected with Pax6 siRNA; Grey circles, values for transfected with both Pax6 and Pcsk1n siRNA; White circles, scrambled siRNA. D, the true insulin and total insulin concentrations in conditional medium of MIN6 cells were measured by ELISA. The cells were transfected with scrambled siRNA, Pax6 siRNA or Pax6 and Pcsk1n siRNA, and aliquots of conditional medium were applied to ELISA Kits for total insulin or true insulin detection (n = 3). True insulin, the mature form of insulin, which was derived from proinsulin cleaved by PC1/3 and PC2; total insulin, the sum of true insulin and proinsulin. White bars, value for transfected with scrambled siRNA; Black bars, value for transfected with Pax6 siRNA; Grey bars, value for transfected with both of Pax6 and Pcsk1n siRNA. A representative experiment is shown; the experiment was repeated three times. Each value represents the mean ± SE. *p<0.05, **p<0.01.