Immunofluorescent detection of protein CoAlation in mammalian cells under oxidative stress

Abstract

Previously, we reported the generation and characterisation of highly specific anti-CoA monoclonal antibodies capable of recognizing CoA in various immunological assays. Utilizing these antibodies in conjunction with mass spectrometry, we identified a wide array of cellular proteins modified by CoA in bacteria and mammalian cells. Furthermore, our findings demonstrated that such modifications could be induced by oxidative or metabolic stress. This study advances the utility of anti-CoA monoclonal antibodies in analysing protein CoAlation, highlighting their effectiveness in immunofluorescent assay. Our data corroborates a significant increase in cellular protein CoAlation induced by oxidative agents. Additionally, we observed that hydrogen-peroxide induced protein CoAlation is predominantly associated with mitochondrial proteins.

Keywords: Anti-CoA mabs; Cell signalling; Coenzyme A; Immunofluorescent analysis; Oxidative stress; Protein CoAlation.

Fig. 1.

IF detection of anti-CoA mAb immunoreactive signal in HEK293 (A) and HEK293/Pank1β (B) cells. Cells were untreated or treated with 0.5 mM H₂O₂ before being fixed with formalin, permeabilised, and processed for immunofluorescence confocal microscopy using anti-CoA mAb (1F10) and FITC-anti-mouse secondary antibodies (green). DAPI (blue) was used to visualise the nuclei. Scale bars: 10 µm.

Fig. 2.

IF detection of anti-CoA mAb immunoreactive signal in MCF7 (A) and MDA-MB-231 (B) cells. Cells were untreated or treated with 0.5 mM H₂O₂ before being fixed with formalin, permeabilised, and processed for immunofluorescence confocal microscopy using anti-CoA mAb (1F10) and FITC-anti-mouse secondary antibodies (green). Nuclei were visualised by DAPI staining (blue). Scale bars: 10 µm.

Fig. 3.

IF detection of the anti-CoA mAb immunoreactive signal in H₂O₂-treated HEK293/Pank1β cells. Formalin-fixed HEK293/Pank1β cells were incubated with primary anti-CoA mAbs (F10) in absence or presence of 10 mM CoA. Following this, immunofluorescence confocal microscopy analysis was performed using FITC-anti-mouse secondary antibodies (green). Scale bars: 10 µm.

Fig. 4.

Effect of DTT treatment of formalin-fixed cells on the intensity of anti-CoA and anti-β tubulin mAbs immunoreactive signals in H₂O₂-stressed HEK293/Pank1β cells. Samples of HEK293/Pank1β formalin-fixed cells prior to incubation with anti-CoA mAbs (F10) (A) or anti-β tubulin mAbs (B) were incubated in PBS supplemented with 200 mM DTT with following immunofluorescence confocal microscopy analysis using FITC-anti-mouse secondary antibodies (green) and nuclear (blue) DAPI staining. Scale bars: 10 µm.

Fig. 5.

Effects of CIAP treatment on H₂O₂-treated MCF7. Formalin-fixed MCF7 cells prior to incubation with anti-CoA mAbs were treated with CIAP following H₂O₂ incubation within the indicated time period. Confocal microscopy analysis was then performed using Cy5-anti-mouse secondary antibodies (green) and nuclear (blue) DAPI staining. Scale bars: 10 µm.

Fig. 6.

Mitochondrial localisation of anti-CoA mAbs immunoreactive signals in H₂O₂ treated HEK293/Pank1β cells (A) with Z-stack imaging of merged anti-CoA and MitoTrackerTM Red CMXRos signals (B). Cells were incubated with MitoTrackerTM Red CMXRos (red) before being fixed, permeabilised, and processed for confocal microscopy with the application of anti-CoA mAbs and FITC-anti-mouse secondary antibodies (green). Yellow in the overlay (merge) images appears where the green fluorescence of anti-CoA mAbs co-localises with the red fluorescence of MitoTracker. DAPI was used to visualise the nuclei (blue). Six representative optical sections with z-step of 1 µm and merged fluorescence signals are shown in B. The samples were imaged with a 63x oil objective lense (NA=1,4) using the Zeiss META 510 confocal microscope. Scale bars: 10 µm.