The PY-type nuclear localization signal (PY-NLS) is required for Gli activation.

<p>(<b>A</b>) Western blot of cell lysates from the indicated cell lines indicated that Myc-Gli2^WT and Myc-Gli2^mPY were expressed at comparable levels that were slightly higher than that of endogenous Gli2. (<b>B–C</b>) Normalized mRNA levels of endogenous <i>Gli1</i> (<b>B</b>) or <i>Patch1</i> (<b>C</b>) measured by quantitative reverse transcription PCR (RT-qPCR) in control (green fluorescent protein [GFP] short hairpin RNA [shRNA]) or Gli2-depeleted NIH3T3 cells with or without lentiviral infection of the indicated Gli2 constructs. (<b>D</b>) Fractionation of Myc-Gli2^WT and Myc-Gli2^mPY from the indicated cell lines treated with or without SAG. Quantification of protein level is shown in the bottom panel. (<b>E–F</b>) Immunostaining (<b>E</b>) and quantification (<b>F</b>) of ciliary-localized Myc-Gli2^WT or Myc-Gli2^mPY in NIH3T3^mGli2-shRNA cells treated with or without sonic hedgehog (Shh). <i>N</i> = 50 cells were examined for each Gli construct. Data are means ± SD from 2 independent experiments. <i>**P < 0</i>.<i>01</i>. (<b>G</b>) <i>Gli-luciferase</i> assay was performed in NIH3T3 cells transfected with the indicated constructs. Data are means ± SD from 2 independent experiments. *<i>P</i> < 0.05, NS: not significant. The underlying data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002063#pbio.2002063.s005" target="_blank">S1 Data</a>.