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. 2012 Mar 14;7(3):e33266. doi: 10.1371/journal.pone.0033266

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Shinde et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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A: Ratios of normalized S334ter-4 Rho BiP, CHOP, pATF6 (90), pATF6 (50), peIF2α ,spliced Xbp1(sXbp1), unsliced Xbp1 (uXbp1), cleaved caspase-12 proteins to corresponding proteins in SD CHOP were used to register the alteration of protein expression. Normalization of all proteins was done by detecting β-Actin protein The ER stress markers BIP protein was a 1.5-fold upregulated in P12 S334ter-4 Rho retinas compared with SD retinas (0.047±0.008 vs. 0.071±0.005, respectively; P = 0.01). In P15, the level of BiP protein was significantly reduced and was not distinguishable from SD. The Chop protein was also dramatically over-expressed (3.5-fold) (0.016±0.005 in SD vs. 0.60±0.002 S334ter-4 Rho, P = 0.0003) on P15 in S334ter-4 Rho retinas. The full length of pAtf6 protein (90 kD) (the Atf6 pathway) was significantly elevated in S334ter-4 Rho retina by 2.7-fold (0.0017±0.0011 in SD vs. 0.0048±0.0007 in S334ter-4 Rho, P = 0.04). The cleaved pAtf6 (50) was significantly elevated by 1.93-fold inS334ter-4 Rho retina (0.18±0.004 in SD vs. 0.035±0.002, P = 0.004). The peIF2α protein was also significantly increased in S334ter-4 Rho retina and was 0.001±0.0005 in S334ter-4 Rho vs 0.002±0.0004, P = 0.0009. The full length of pAtf6 protein (90 kD) (the Atf6 pathway) was significantly elevated in S334ter-4 Rho retina by 2.7-fold (0.0017±0.0011 in SD vs. 0.0048±0.0007 in S334ter-4 Rho, P = 0.04). The N-terminal of the full-length of Atf6, cleaved pAtf6 was elevated by 1.93-fold and was 0.18±0.004 in SD vs. 0.035±0.002, P = 0.004. We also observed that the peIF2α protein was significantly increased in S334ter-4 Rho retina and was 0.001±0.0005 in S334ter-4 Rho vs 0.002±0.0004, P = 0.0009. The spliced Xbp1 protein was detected in S334ter-4 retina. Its level was a 4.5-fold higher in transgenic retina compared to SD and was 0.022±0.003 in SD and 0.1±0.01, P = 0.001 in S334ter-4 rats. Unspliced Xbp1 protein was increased to a lesser extent (2.3-fold) in S334ter-4 rats and was 0.049±0.008 in SD and 0.13±0.001, P = 0.034 in S334ter-4. Increase in active caspase-12 was observed in S334ter-4 Rho retina. The level of cleaved caspase-12 (20 kD) was elevated in S334ter-4 Rho rats on P15 compared to control over 2-fold and was 0.05±0.007 in SD vs. 0.12±0.02 in S334ter-4 Rho, P = 0.014 on P15. B: Upper panel: Quantification of spliced form of the Xbp1 mRNA (the IRE signaling) detected by RT-PCR reaction. We observed 1.45-fold increased in the spliced form of Xbp1 mRNA in S334ter-4 Rho retina. The ratio of spliced Xbp1 to normalized unspliced Xbp1 mRNA was 0.22±0.0006 in SD vs. 0.033±0.031 in S334ter-4 Rho retina, P = 0.025). Image of the agarose gel loaded with RT-PCR product obtained with Xbp1 specific primers is shown in a lower panel. C: Images of western blots treated with anti-Actin, Bip, CHOP, peIf2α, pATF6, Xbp1 antibodies and detected with secondary antibodies and infrared imaging scanner are presented.