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. 2019 Nov 7;101(3):507–517. doi: 10.1111/tpj.14556

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© 2019 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd

This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

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UVR8‐mediated degradation of PIF4 and PIF5 is associated with reduced target promoter occupancy in response to UV‐B.

(a)–(c) Anti‐hemagglutinin (HA) immunoblot analysis of HA‐tagged proteins in 4‐day‐old seedlings grown under white light (−UV‐B) or white light supplemented with a final 6 h of UV‐B (+UV‐B). The wild type (Col) is shown as a negative control. PIF4‐HA protein levels (a) were analyzed in Col/Pro_PIF4:PIF4‐3×HA (PIF4‐HA) and uvr8‐6/Pro_PIF4:PIF4‐3×HA (uvr8/PIF4‐HA). PIF5‐HA protein levels (b) were analyzed in Col/Pro_PIF5:PIF5‐3×HA (PIF5‐HA) and uvr8‐6/Pro_PIF5:PIF5‐3×HA (uvr8/PIF5‐HA). PIF4‐HA protein levels (c) were analyzed in Col/Pro_PIF4:PIF4‐3×HA (PIF4‐HA) and cop1‐4/Pro_PIF4:PIF4‐3×HA (cop1‐4/PIF4‐HA). Blots were reprobed with anti‐UVR8 (a,b), as well as anti‐actin as loading control (a–c).

(d) Chromatin immunopreciptation (ChIP) of DNA associated with PIF5‐HA. Chromatin immunopreciptation‐qPCR was performed for the XTR7 and IAA19 promoters and an intergenic region between the At4g26900 and At4g26910 genes using 5‐day‐old Col/Pro_PIF5:PIF5‐3×HA (PIF5‐HA) seedlings either exposed to narrowband UV‐B for 6 h (WL + UVB) or not (WL), and wild‐type (Col; negative control) seedlings grown only under white light (WL).

(e) Chromatin immunopreciptation of DNA associated with PIF4‐HA. Chromatin immunopreciptation‐qPCR was performed for the XTR7 promoter using 5‐day‐old Col/Pro_PIF4:PIF4‐3×HA (PIF4‐HA) seedlings either exposed to narrowband UV‐B for 6 h (WT + UVB) or not (WL) and wild‐type (Col; negative control) seedlings grown only under white light (WL). In (d) and (e) the ChIP experiments were performed with an anti‐HA antibody. The numbers of the analyzed DNA fragments (pro_{XTR7_‐1972}, pro_{IAA19_‐335}) indicate the positions of the base pair of the amplicon relative to the translation start site (referred to as position +1). The percentage of DNA associated with (d) PIF5‐HA and (e) PIF4‐HA relative to total input DNA of two independent biological replicates were normalized against the PIF5‐HA WL and PIF4‐HA WL sample, respectively (NormVal, PIF5‐HA WL = 1 and PIF4‐HA WL = 1). Means are represented as horizontal bars. P‐values from pairwise t‐tests using Holm's correction for multiple tests are shown.