RESUMO
Primary antibodies are one of the main tools used in molecular biology research. However, the often-occurring cross-reactivity of primary antibodies complicates accurate data analysis. Our results show that three commercial polyclonal antibodies raised against N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) strongly cross-react with endogenous and recombinant mitosis-associated protein Aurora kinase A (AURKA). The cross-reactivity was verified through immunofluorescence, immunoblot, and immunoprecipitation assays combined with mass spectrometry. N6AMT1 and AURKA are evolutionarily conserved proteins that are vital for cellular processes. Both proteins share the motif ENNPEE, which is unique to only these two proteins. We suggest that N6AMT1 antibodies recognise this motif in N6AMT1 and AURKA proteins and exhibit an example of "specific" non-specificity. This serves as an example of the importance of controls and critical data interpretation in molecular biology research.
RESUMO
The methyltransferase N6AMT1 has been associated with the progression of different pathological conditions, such as tumours and neurological malfunctions, but the underlying mechanism is not fully understood. Analysis of N6AMT1-depleted cells revealed that N6AMT1 is involved in the cell cycle and cell proliferation. In N6AMT1-depleted cells, the cell doubling time was increased, and cell progression out of mitosis and the G0/G1 and S phases was disrupted. It was discovered that in N6AMT1-depleted cells, the transcription of cyclin E was downregulated, which indicates that N6AMT1 is involved in the regulation of cyclin E transcription. Understanding the functions and importance of N6AMT1 in cell proliferation and cell cycle regulation is essential for developing treatments and strategies to control diseases that are associated with N6AMT1.
