IRE1α-mediated unconventional splicing of XBP1 is emerging as a biomarker in several disease states and is indicative of activation of the unfolded protein response sensor IRE1. Splicing of XBP1 mRNA results in the translation of two distinct XBP1 protein isoforms (XBP1s and XBP1u) which, due to post-translational regulation, do not correlate with mRNA levels. As both XBP1 isoforms are implicated in pathogenic or disease progression mechanisms there is a need for a reliable, clinically applicable method to detect them. A multiplexed isoform-specific XBP1 array utilising Biochip array technology (BAT™) was assessed for specificity and suitability when using cell protein lysates. The array was applied to RIPA protein lysates from several relevant pre-clinical models with an aim to quantify XBP1 isoforms in comparison with RT-PCR or immunoblot reference methods. A novel reliable, specific and sensitive XBP1 biochip was successfully utilised in pre-clinical research. Application of this biochip to detect XBP1 splicing at the protein level in relevant breast cancer models, under basal conditions as well as pharmacological inhibition and paclitaxel induction, confirmed the findings of previous studies. The biochip was also applied to non-adherent cells and used to quantify changes in the XBP1 isoforms upon activation of the NLRP3 inflammasome. The XBP1 biochip enables isoform specific quantification of protein level changes upon activation and inhibition of IRE1α RNase activity, using a routine clinical methodology. As such it provides a research tool and potential clinical tool with a quantified, simultaneous, rapid output that is not available from any other published method.

中文翻译：

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一种新的多路复用阵列在拼接和未拼接 XBP1 的快速、灵敏、同步和定量评估中的应用

IRE1α 介导的 XBP1 非常规剪接正在成为几种疾病状态的生物标志物，并表明未折叠蛋白反应传感器 IRE1 的激活。XBP1 mRNA 的剪接导致两种不同的 XBP1 蛋白同种型（XBP1s 和 XBP1u）的翻译，由于翻译后调节，它们与 mRNA 水平不相关。由于两种 XBP1 同种型都与致病或疾病进展机制有关，因此需要一种可靠的、临床适用的方法来检测它们。使用生物芯片阵列技术 (BAT™) 评估了多重异构体特异性 XBP1 阵列在使用细胞蛋白裂解物时的特异性和适用性。该阵列被应用于来自几个相关临床前模型的 RIPA 蛋白裂解物，旨在与 RT-PCR 或免疫印迹参考方法相比量化 XBP1 同种型。一种新型可靠、特异性和灵敏的 XBP1 生物芯片已成功用于临床前研究。在基础条件下以及药理抑制和紫杉醇诱导下，将该生物芯片应用于检测相关乳腺癌模型中蛋白质水平的 XBP1 剪接，证实了先前研究的结果。该生物芯片还应用于非贴壁细胞，并用于量化 NLRP3 炎性体激活后 XBP1 同种型的变化。XBP1 生物芯片能够使用常规临床方法对激活和抑制 IRE1α RNase 活性后的蛋白质水平变化进行异构体特异性量化。