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. 2021 Mar 11;11:642271. doi: 10.3389/fcimb.2021.642271

Figure 2.

Figure 2

Over-expression of ATAT-HA in epimastigotes increases α-tubulin acetylation. (A) Immunolocalization of ATAT-HA with rat monoclonal anti-HA antibodies in Dm28c pTcINDEX-GW ATAT-HA epimastigotes induced with 0.5 μg/ml tetracycline for 24 h. Bar: 5 μm. DAPI was used as nucleus and kinetoplast marker. The light blue arrow indicates the kinetoplast and the pink arrow indicates the nucleus. (B) Total extracts of pTcINDEX-GW ATAT-HA epimastigotes in the absence (-) or presence (+) of 0.5 μg/ml tetracycline for 24 h were separated by SDS/PAGE and stained with Coomassie Blue (left panel), followed by Western blot analysis using rat monoclonal anti-HA antibodies (right panel). (C) Western blot of total extracts of pTcINDEX-GW ATAT-HA epimastigotes with 0.5 μg/ml tetracycline a different time points post-induction using rat monoclonal anti-HA, mouse monoclonal anti-acetylated a-tubulin (anti-AcTub), rabbit monoclonal anti-TcATAT antibodies and mouse monoclonal anti-a-tubulin (anti- αTub). Bands were quantified by densitometry using α-tubulin signal to normalize the amount of ATAT-HA and acetylated α-tubulin (right panel). Plots represent one of three independent experiments performed. (D) ATAT-HA autoacetylation assay. ATAT-HA was purified from T. cruzi epimastigotes and incubated in the absence (-) and presence (+) of AcetylCoA and then separated by SDS/PAGE followed by Western blot analysis with rat monoclonal anti-HA antibodies and rabbit monoclonal anti-Acetylated Lysine (anti-AcLys).