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. 2022 Aug 13;56:102431. doi: 10.1016/j.redox.2022.102431

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© 2022 The Authors. Published by Elsevier B.V.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Fig. 1 — LIX1 localizes to mitochondria and is tightly anchored to the outer membrane.

(A) Confocal microscopy analysis of HA-LIX1 localization in HeLa cells. Comparison with the mitochondrial marker COXIV. Scale bars, 10 μm. (B) RGB profile of LIX1 and COXIV expression co-localization. (C) Western blot analysis of total protein extracts (Total), crude cytoplasmic (Crude Cyto) and purified mitochondria (Purified Mito) from non-transfected (Empty) and HA-LIX1-expressing HeLa cells. Membranes were probed with antibodies against the HA tag, GAPDH (cytoplasmic) and TFAM (mitochondrial marker). (D) Sub-mitochondrial localization of HA-LIX1 in isolated mitochondria from HA-LIX1-expressing HeLa cells after (+) or not (−) proteinase K (PK) incubation (accessibility test). Membranes were probed with antibodies against the HA tag, TOM20 and VDAC (Outer Mitochondrial Membrane, OMM, markers), COXIV (Inner Mitochondrial Membrane, IMM, marker), Cytochrome C (CYT C; Inter-Membrane Space, IMS, marker), and TFAM (matrix protein). (E) Western blot analysis of total isolated mitochondria (Mito) from HA-LIX1-expressing cells after incubation with Na₂CO₃ pH 11.5 and ultracentrifugation to separate the supernatant (S) and pellet (P) fractions. Membranes were probed with antibodies against COXIV (IMM marker) and cytochrome C (CYT C) (IMS soluble marker). (F) Western blot analysis of total isolated mitochondria (Mito) from HA-LIX1-expressing HeLa cells after solubilization (+) or not (−) with the indicated detergents and ultracentrifugation. Membranes were probed with antibodies against the HA tag, TFAM (matrix protein), VDAC and TOM20 (OMM markers), and COXIV (IMM marker).