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The authors would like to clarify several issues recently raised by the PLOS Biology editors.
In the legend for Fig 1, it was not made clear that the E988K lane in panel C is the same as in Fig 6B. The authors have provided a corrected legend for Fig 1, which now states the origin of the E988K lane in panel C.
In the legend for Fig 2C, it was not made clear that these data are being re-used from Fig 1C. The authors have provided a corrected legend for Fig 2, which now states the origin of the data shown in Fig 2C.
The published version of Fig 7C was constructed from original data shown in Figure 3B and 6B. The lanes 4–6 (noted as ggAID o/e) of Figure 3B are the source of lanes 1, 2, and 6 in the Fig 7C panel. The lanes 1–3 in Fig 6B are the source of the data for lanes 3, 4, and 5 in the Fig 7C panel. The composite panel was submitted without appropriate annotations in Fig 7C in error.
As requested by the editors, the authors have re-run this experiment, using the same cell lines as used in the original experiments and, with this newly generated data, provided a corrected version of the figure here. The editors have verified the data underlying the corrected panel and are satisfied that these continue to uphold the conclusions from this figure. Note that the figure legend remains unchanged.
Methods:
Frozen aliquots of the cells used in the original experiments were expanded, lysed, and western blots for PARP, AID, and β-actin (as loading control) were generated. “Start of culture” cells were expanded for 4 days in culture, resulting in a total of c.a. 14 days expansion from a single clone. “End of culture” cells were expanded an additional 8 days for a total of c.a. 31 days total expansion. The resulting time differential approximately replicates the state of the cells analyzed in the original Fig 7C.
For each sample, protein content was quantitated, and Western blotting was performed using the methodology described in the paper. Secondary-antibody only blots were performed to assess for potential for non-specific bands related to the secondary antibody, and no bands were detected. Primary antibodies used for detection of specific proteins were 4C10-5 anti-PARP (bdbiosciences, catalog #556494), Anti-AICDA LS-C34861 (LS Biosciences), and anti-β–actin clone AC-74 (Sigma Aldrich). Western blot images were obtained on a Licor Odyssey Imaging System (Licor.com), and contrast-adjusted for illustrative purposes.
For compatibility with the original panel 7C legend, fragments corresponding to the “end of culture period” and “start of culture period” were excised from images of respective specific protein western blots in which all samples were processed and analyzed simultaneously.