Xenbase Image ID: 139009

FIGURE 2. Hspa5MO specifically knocks down Hspa5 in Xenopus embryos. A, the two Hspa5MOs targeted to the ATG sequence are depicted in blue font and underlined.Thetranslation start site ofhspa5isshownin black with theATGin red.B–E,Hspa5MO1and-MO2reducedendogenousHspa5protein inHspa5morphants. Xenopus embryos at the two-cell stage were injected with the indicated doses of Hspa5MO1 (B) or Hspa5MO2 (D) and collected at stage 11. Endogenous Hspa5 was detected by Western blotting using -Hspa5 antibody. Injection of Hspa5MO1 reduced endogenous Hspa5 in a dose-dependent manner in Xenopus embryos. The knockdown experiments were repeated twice for each MO, and a representative gel is shown in B and D. Western blots shown in B and D were quantified in C and E, respectively. The signal densities were acquired and analyzed using a GS-800 calibrated imaging densitometer. The relative signals of Hspa5 to tubulin from either Hspa5MO1- or Hspa5MO2-injected embryos were normalized to those from control embryos. The ratios indicate the decrease of Hspa5 expression. F,Hspa5MO1and Hspa5MO2 induced the same phenotypes. The injected embryos show a range of phenotypes of short and curved body axis and disruption of eye and other head structures. The phenotypes of embryos injected with Hspa5MO1 or -MO2 were categorized into mild, moderate, and severe groups. G, quantification of phenotypes thatwereinducedbyincreasing doses ofHspa5MO1orHspa5MO2.Thenumbersabovethecolumnsindicate thenumberof injectedembryosfromthree experiments, and the phenotypes were categorized by investigators who were blinded to the treatment. H, silent mutations were introduced into the pCS2-hspa5 sequence to prevent binding of Hspa5MOs. I, the pCS2-hspa5 mRNA partially rescued the phenotype induced by Hspa5MOs. Quantification of phenotypes induced by the indicatedHspa5MOsoracombinationofpCS2-hspa5mRNAandHspa5MOisshownin thecolumnchart. Threeindependentexperimentswereperformed,andhspa5 mRNA partially rescued the phenotype induced by Hspa5MO1. The number of injected embryos is listed on the top of the columns. Image published in: Shi W et al. (2015) Copyright © 2015. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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