Journal: bioRxiv

Article Title: Proteomic analysis of the role of the quality control protease LONP1 in mitochondrial protein aggregation

doi: 10.1101/2021.04.12.439502

Figure Lengend Snippet: Detection of different mitochondrial and cytosolic proteins in total cell lysates (A) and isolated mitochondria (B) of WT and LONP1 gKD cells by SDS-PAGE, Western blot and immunodetection. Antibodies against the indicated proteins were used as listed in Material and Methods. Indicated are the corresponding relative molar masses of the respective SDS-PAGE marker proteins. GAPDH and HSP60 signals were used as loading control. (C) LONP1 protein levels (given as ratio to WT) in isolated mitochondria of WT (black) and LONP1 gKD (gray) cells used for quantitative MS analysis after two weeks labelling with normal or heavy isotope-labelled arginine. (D) Determination of ATP concentration in isolated and lysed WT (black) or LONP1 gKD (gray) mitochondria. ATP concentration was determined by using a luciferase-based assay as described. Shown are mean values ± SEM of three independent experiments (n=3), ** p<0.01. (E) Detection of mitochondrial inner membrane potential (Δψ) in isolated mitochondria of WT (black) and LONP1 gKD (dark gray) cells, using the potential-dependent fluorescent dye TMRE. As control, mitochondria were pre-treated with 1 μM valinomycin for 10 min at room temperature. Shown are mean values ± SEM (n=3) of TMRE fluorescence emission intensities. (F) Measurement of mitochondrial ROS amounts in WT (black) and LONP1 gKD (dark gray) cells by flow cytometry analysis using the mitochondria-specific superoxide dye MitoSOX. As positive control, cells were pre-treated with 0.1 mM menadione (Me) for 60 min (WT, gray; LONP1 gKD, bright gray). Shown are mean values ± SEM (n=3) of MitoSOX fluorescence emission intensity; ** p<0.01. (G) Immunofluorescence analysis of mitochondrial morphology by using fluorescence microscopy of fixed WT and LONP1 gKD cells. Anti-SDHA antibodies were used to visualize the mitochondrial network (red), nuclei were stained by DAPI (blue). Upper panel, HeLa WT cells; lower panel, LONP1 gKD cells. Bar: 50 μm.

Article Snippet: Samples were loaded onto an 12.5% SDS-PAGE and analyzed by Western blot and immunodecoration with αACO2 (Sigma Aldrich HP001097), αLONP1 (own work), αHSP60 (Santa Cruz 13966), αTUFM Protein (Tech 11701-1-AP), αTSFM (Sigma Aldrich HPA024087), αTFAM (NEB 80760S) and αTIMM23 (BD.

Techniques: Isolation, SDS Page, Western Blot, Immunodetection, Marker, Control, Concentration Assay, Luciferase, Membrane, Fluorescence, Flow Cytometry, Positive Control, Immunofluorescence, Microscopy, Staining