Division of Molecular and Cellular Pathology, Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, United States.
Am J Physiol Heart Circ Physiol. 2023 Oct 1;325(4):H601-H616. doi: 10.1152/ajpheart.00104.2023. Epub 2023 Aug 4.
The modification of serine and threonine amino acids of proteins by -linked -acetylglucosamine (-GlcNAc) regulates the activity, stability, function, and subcellular localization of proteins. Dysregulation of -GlcNAc homeostasis is well established as a hallmark of various cardiac diseases, including cardiac hypertrophy, heart failure, complications associated with diabetes, and responses to acute injuries such as oxidative stress and ischemia-reperfusion. Given the limited availability of site-specific -GlcNAc antibodies, studies of changes in -GlcNAcylation in the heart frequently use pan--GlcNAc antibodies for semiquantitative evaluation of overall -GlcNAc levels. However, there is a high degree of variability in many published cardiac -GlcNAc blots. For example, many blots often have regions that lack -GlcNAc positive staining of proteins either below 50 or above 100 kDa. In some -GlcNAc blots, only a few protein bands are detected, while in others, intense bands around 75 kDa dominate the gel due to nonspecific IgM band staining, making it difficult to visualize less intense bands. Therefore, the goal of this study was to develop a modifiable protocol that optimizes -GlcNAc positive banding of proteins in cardiac tissue extracts. We showed that -GlcNAc blots using CTD110.6 antibody of proteins ranging from <30 to ∼450 kDa could be obtained while also limiting nonspecific staining. We also show that some myofilament proteins are recognized by the CTD110.6 antibody. Therefore, by protocol optimization using the widely available CTD110.6 antibody, we found that it is possible to obtain pan--GlcNAc blots of cardiac tissue, which minimizes common limitations associated with this technique. The post-translational modification of proteins by -linked -acetylglucosamine (-GlcNAc) is recognized as mediating cardiac pathophysiology. However, there is considerable variability in the quality of -GlcNAc immunoblots used to evaluate changes in cardiac -GlcNAc levels. Here we show that with relatively minor changes to a commonly used protocol it is possible to minimize the intensity of nonspecific bands while also reproducibly generating -GlcNAc immunoblots covering a range of molecular weights from <30 to ∼450 kDa.
蛋白质丝氨酸和苏氨酸残基的 - 连接 -N- 乙酰葡萄糖胺（-GlcNAc）修饰调节蛋白质的活性、稳定性、功能和亚细胞定位。-GlcNAc 动态平衡失调已被确定为各种心脏疾病的标志，包括心肌肥厚、心力衰竭、与糖尿病相关的并发症以及对氧化应激和缺血再灌注等急性损伤的反应。鉴于特异性 -GlcNAc 抗体的可用性有限，因此，研究心脏中 -GlcNAc 酰化的变化通常使用泛 -GlcNAc 抗体对半定量评估整体 -GlcNAc 水平。然而，许多已发表的心脏 -GlcNAc 印迹中存在高度的可变性。例如，许多印迹通常有蛋白质缺乏 -GlcNAc 阳性染色的区域，要么低于 50 kDa，要么高于 100 kDa。在一些 -GlcNAc 印迹中，只有少数蛋白质带被检测到，而在其他印迹中，由于非特异性 IgM 带染色，75 kDa 左右的强烈带主导凝胶，使得较不强烈的带难以可视化。因此，本研究的目的是开发一种可修改的方案，以优化心脏组织提取物中蛋白质的 -GlcNAc 阳性带。我们表明，可以获得范围从 <30 到 ∼450 kDa 的蛋白质的 CTD110.6 抗体 -GlcNAc 印迹，同时限制非特异性染色。我们还表明，一些肌丝蛋白被 CTD110.6 抗体识别。因此，通过使用广泛可用的 CTD110.6 抗体优化方案，我们发现可以获得心脏组织的泛 -GlcNAc 印迹，最大限度地减少了与该技术相关的常见限制。蛋白质的 - 连接 -N- 乙酰葡萄糖胺（-GlcNAc）的翻译后修饰被认为介导心脏病理生理学。然而，用于评估心脏 -GlcNAc 水平变化的 -GlcNAc 免疫印迹的质量存在相当大的差异。在这里，我们表明，通过对常用方案进行相对较小的改变，可以最大限度地减少非特异性带的强度，同时还可以可重复地生成覆盖 <30 到 ∼450 kDa 分子量范围的 -GlcNAc 免疫印迹。
