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. Author manuscript; available in PMC: 2016 Dec 27.
Published in final edited form as: J Immunol. 2015 Oct 5;195(10):4760–4770. doi: 10.4049/jimmunol.1500979

FIGURE 7.

FIGURE 7

Effect of UNC-45A knockdown on NMIIA expression levels and ATP-sensitive interactions of NMIIA with actin filaments. (A) Expression levels of UNC-45A, NMIIA, pNMIIA H chain (HC), and pNMIIA L chain (LC) in YT cells transduced with either shRNA-scramble or shRNA targeting two different UNC-45A regions. β-Actin was used as a loading control. (B) NMIIA-actin binding capability in YT cells transduced with either scramble or shRNA targeting two different UNC-45A regions in the absence (−) and presence (+) of the ATP diphosphohydrolase apyrase. (C) NMIIA-actin binding capability in YT cells transduced with empty vector (lenti-pEF-GFP) or lenti-pEF-UNC-45A–GFP in the absence (−) and presence (+) of the ATP diphosphohydrolase apyrase. (D) Quantification of NMIIA actin-binding capability expressed as a percentage of the control (average of two independent experiments) and obtained by comparing lines 5 and 7 and 9 and 11 of (B) and lines 4 and 6 of (C). (E) YT NK cells expressing UNC-45A–GFP fusion protein were incubated with 721.221 target cells. After fixation, effector-target conjugates were stained with an anti-NMIIA polyclonal Ab and an anti-actin mAb followed by Texas red goat anti-rabbit IgG and Alexa Fluor 647-conjugated donkey anti-mouse IgG and analyzed by fluorescence microscopy. Arrow indicates the NKIS.