To investigate a direct interaction between the compound and the protein, we expressed and purified recombinant AIFM1. The protein was spiked in HEK-293 lysate followed by addition of 5 µM Cipro P1, click to rhodamine azide and analysis via fluorescent SDS-PAGE analysis. A strong fluorescent band corresponding to the molecular weight of AIFM1 was visible in the spiked sample, which was absent in the control lacking the protein (Figure ). Moreover, addition of excess Ciprofloxacin prior to probe addition resulted in a strong reduction of signal intensity, confirming that Ciprofloxacin binds AIFM1 and addresses the same binding site as the probe. Consequently, our results indicate that Ciprofloxacin binds AIFM1, thereby inducing mitochondrial dysfunction and ETC complex I and IV downregulation. showing the comparison of respiratory chain complex protein levels in HEK293 WT or AIFM1 KO cells treated with ciprofloxacin or left untreated. Changes of protein levels of all significantly affected subunits of the respective respiratory chain complexes upon ciprofloxacin treatment were plotted. In WT cells, levels of complex I and IV subunits decrease upon ciprofloxacin treatment. This effect is attenuated in AIFM1 KO cells indicating that ciprofloxacin acts on AIFM1.