Myosin IIA is constitutively phosphorylated at S1943. (A-D) YTS cells expressing wild-type myosin IIA-GFP (MIIA) or 1933x myosin IIA-GFP (1933x) were left resting or were conjugated to KT86 target cells. GFP-tagged proteins were immunoprecipitated from lysates (A,C) or from the supernatants of isolated lytic granules that were treated with NaCl to remove surface proteins (B,D). GFP immunoprecipitates were evaluated by gel staining for phosphorylation (Pro-Q) and total protein (Sypro) (A-B), or by Western blot for phospho-S1943 and total myosin IIA (C-D). In all gels of GFP immunoprecipitates, lanes were loaded for equal GFP-myosin content to facilitate comparison of phosphorylation, and therefore the 1933x lanes contain greater total protein. In panels A through D, numbers below blots indicate the ratio of the phosphorylated band intensity to the total protein intensity for the GFP-tagged protein (upper band) normalized to the ratio for myosin IIA. In each image, there are 2 myosin bands: the larger corresponds to 253 kDa and represents the myosin IIA GFP fusion, which is enriched in the immunoprecipitates; the smaller corresponds to 227 kDa and represents the endogenous myosin IIA. The inclusion of 2 heavy chains in each myosin hexamer enables association of exogenous and endogenously expressed myosin IIA molecules when evaluated via immunoprecipitation (see also supplemental Figure 1). (E) Resting and stimulated ex vivo human NK-cell lysates and lytic granules isolated from resting eNK cells evaluated by Western blot for phospho-S1943 and total myosin IIA. Results are representative of 3 (A-E, eNK) or 2 (E, LG) independent assays.