Par3-OGDH相互作用定位验证：线粒体共IP方法学挑战与解决方案

The subcellular compartment where the Par3–OGDH interaction occurs is not directly demonstrated by fraction-restricted assays. Although colocalization (Figure 2C) and PLA (Figure 2D, Figure S2A) suggest proximity near mitochondria, neither establishes that binding occurs within mitochondria rather than in mixed cellular lysates. The co-IPs (Figure 2E–2F) appear to be from whole-cell extracts, which cannot exclude cytosolic or non-matrix interactions. This concern is supported by evidence that OGDH-like proteins can exhibit unexpected subcellular localizations, including nuclear rather than mitochondrial localization [1]. Matrix-restricted co-IP or protease-protected mitochondrial co-IP would address this gap.