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. 2023 Feb 16;14:1063368. doi: 10.3389/fmicb.2023.1063368

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Copyright © 2023 Lara-Ochoa, Huerta-Saquero, Medrano-López, Deng, Finlay, Martínez-Laguna and Puente.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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GrlR and GrlA are expressed under both repressing and inducing conditions and interact to form the GrlR₂-GrlA heterotrimer. The enteropathogenic Escherichia coli (EPEC) grlR:3xFLAG (A) and grlA:3xFLAG (B) strains were grown in 50 ml of Dulbecco’s modified Eagle’s medium (DMEM) or Lysogeny Broth (LB) with shaking at 37°C. Total cell extracts were collected at OD₆₀₀ of 0.3, 0.6, 0.8, and 1.0 and separated by 12% native PAGE. GrlR₂-GrlA and GrlR-GrlR complexes and GrlR monomer are indicated by arrows and were identified by western blotting using anti-FLAG monoclonal antibodies. The same cell extracts were also separated by 12% SDS-PAGE to evaluate the expression of GrlR-FLAG and GrlA-FLAG by western blotting using anti-FLAG antibodies. As a control for protein loading, GroEL expression was also analyzed using anti-GroEL antibodies.