NM IIA2 reduces the actin-gliding velocity of NM IIA.A and B, in vitro motility assay with HMM IIA (A) and NM IIA (B), in which a fixed amount of inserted isoform (HMM IIA2 or NM IIA2) was mixed with varying amount of their corresponding noninserted isoform (HMM IIA0 or NM IIA0) at 1:1, 1:2, or 1:4 ratios, as indicated, in the presence of MLCK. In vitro motility of noninserted isoforms HMM IIA0 and NM IIA0 only were considered as positive controls for A and B, respectively. Data are shown as mean ± SD from n = 405 (HMM IIA0), 95 (1:1), 167 (1:2), 36 (1:4), 306 (NM IIA0), 55 (1:1), 54 (1:2), 72 (1:4) actin filaments from three biological replicates. Blue horizontal line denotes the median value. Data were analyzed using the one-way ANOVA test followed by pairwise student t test. p > 0.05 was considered to be nonsignificant. HMM, heavy meromyosin; MLCK, myosin light chain kinase; NM, nonmuscle myosin.