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. 2017 Jan 30;13(1):e1006131. doi: 10.1371/journal.ppat.1006131

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© 2017 Day et al

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Fig 4 — (A) PTP2 and PTP3 are induced in ypd1Δ cells. Northern blot analysis of PTP2 and PTP3 expression in exponentially growing Wt (JC21 and ypd1Δ (JC2001) cells. ACT1 was used as a loading control. (B) The kinetics of PTP3 and PTP2 are similar to that of Hog1 activation following doxycycline treatment of tetO-YPD1 cells. Northern blot analyses of PTP2 and PTP3 expression, and western blot analysis of Hog1 phosphorylation in tetO-YPD1 cells (JC1586) following treatment with doxycycline for the indicated times. (C) Quantification of PTP3 and PTP2 induction following doxycycline treatment of tetO-YPD1 cells. (D) The tyrosine phosphatase inhibitor, arsenite, further activates Hog1 in ypd1Δ cells. Western blot analysis of whole cell extracts isolated from exponentially growing Wt and ypd1Δ cells treated with 5mM NaAsO₂ for the specified times. Duplicate blots were probed for phosphorylated Hog1 (Hog1-P) or total Hog1 (Hog1) levels. A darker exposure of the Hog1-P blot is included (middle panel) to show the level of Hog1-P observed in Wt cells following arsenite treatment. (E) Hog1 is activated by arsenite in PBS2^DD cells. Western blot analysis of whole cell extracts isolated from exponentially growing PBS2 (JC112), PBS2^AA (JC126) and PBS2^DD (JC124) cells after treatment with 5mM NaAsO₂ for the specified times. Blots were probed for phosphorylated Hog1 (Hog1-P), stripped, and reprobed for total Hog1 (Hog1) levels. (F) Deletion of PTP genes trigger greater activation of Hog1 following repression of YPD1. Western blot analysis of whole cell extracts isolated from tetO-YPD1, tetO-YPD1 ptp3Δ (JC2188) and tetO-YPD1 ptp3Δ PTP2/ptp2 (JC2195) cells following treatment with doxycycline for the indicated times. Duplicate blots were probed for phosphorylated Hog1 (Hog1-P) or total Hog1 (Hog1) levels. (G) Deletion of PTP genes impairs cell growth following repression of YPD1. 10⁴ cells, and 10-fold dilutions thereof, of the indicated strains were spotted onto rich media plates plus or minus DOX, and incubated at 30°C for 24h.