UNC-45a depletion predisposes nascent NM-II for aggregation. (A) Relative protein levels of NM-IIA, NM-IIB, and pp(T18, S19)–RLC were analyzed by WB from WT and UNC-45a knockout (KO) cell lysates. The NM-IIA/B and pp-RLC levels in WT cells were set to 1, and the data are presented as means ± SEM, n = 3 WT and 3 UNC-45a knockout total cell lysates. (B) Quantification of MYH9 (NM-IIA) and MYH10 (NM-IIB) mRNA levels in WT and UNC-45a knockout cells. GAPDH was used as the control to derive the ΔCt values. The data are presented as 2^−ΔΔCt ± SEM, n = 4 (mRNAs extracted from 4 WT and 4 UNC-45a knockout cultures, converted to cDNAs, and analyzed by quantitative PCR). (C) WT and UNC-45a knockout cells were treated for 4 h with 10 µM MG-132 proteasome inhibitor. Yellow arrowheads indicate examples of NM-IIA and NM-IIB containing aggregates in UNC-45a knockout cells. Bars, 20 µm. Fig. S4 A shows a similar analysis of other UNC-45a knockout cell clones obtained. (D) Treatment of WT and UNC-45a knockout cells with a combination of the protein synthesis inhibitor cycloheximide (0.1 mg/ml) and MG-132 (10 µM) for 4 h. Bars, 20 µm. (C and D) F-actin was visualized with phalloidin. NM-IIA and NM-IIB heavy chains and nuclei were detected with polyclonal antibodies and DAPI, respectively.