Immunoblot analysis of NMHCII-A expression levels in adult mouse lung tissue. (A) Analysis of NMHCII-A expression from extracts of A⁺/A⁺ and A^gfpR702C/A⁺ mouse lung. Note that mutant GFP–NMHCII-A protein migrates more slowly than endogenous NMHCII-A. Mutant GFP–NMHCII-A and endogenous NMHCII-A were detected with anti–NMHCII-A. In the A^gfpR702C/A⁺ lung tissue, the expression level of the GFP–NMHCII-A R702C is ∼ 80% of the endogenous NMHCII-A. There are 2 different loadings for each sample. (B) Immunoblot analysis of NMHCII-A expression in A⁺/A⁺, A^D1424N/A⁺, and A^D1424N/A^D1424N tissue shows that approximately the same amount of NMHCII-A protein, normalized to tubulin, is expressed in the A⁺/A⁺, A^D1424N/A⁺, and A^D1424N/A^D1424N mouse lung. (C) Immunoblot analysis of NMHCII-A expression in A⁺/A⁺, A^E1841K/A⁺, and A^E1841K/A^E1841K lung shows that similar amounts of NMHCII-A are expressed normalized to tubulin. (D) Immunoblot analysis of NMHCII-A expression in A^gfp/A⁺ lungs also shows similar expression of GFP–NMHCII-A compared with NMHCII-A. (E) Evidence for heterodimer formation in A^gfpR702C/A⁺ mouse tissue. High-salt lysates of A^gfpR702C/A⁺ lung tissue were incubated with anti-GFP, and the immunoprecipitate was subject to SDS-PAGE followed by immunoblotting with NMHCII-A Ab. Lane 1 indicates A⁺/A⁺ lysate immunoprecipitated with anti-GFP shows no immunoprecipitate; lane 2, A^gfpR702C/A⁺ lysate treated with beads without Ab shows no immunoprecipitate; lane 3, A^gfpR702C/A⁺ lysate immunoprecipitated with anti-GFP shows a 4-fold increase in GFP–NMHCII-A R702C compared with NMHCII-A; and lane 4, A^gfpR702C/A⁺ lysate input showing endogenous NMHCII-A (bottom band) and GFP–NMHCII-A R702C (top band). The signal intensity ratio between the top band and bottom band of lane 3 is 4.09 ± 0.69 (n = 3), ratio for lane 4 is 0.81 ± 0.13 (n = 3). Relative intensity of signal was determined with ImageJ (NIH). (F,G) Immunoprecipitation from A^gfp/A⁺ lung (F, lane 2) and leukocytes (G, lane 2) shows 5.25 ± 0.35 (n = 2) and 3.94 (n = 1) ratio of GFP–NMHCII-A to NMHCII-A, respectively. Lane 1 is immunoprecipitation with beads but without Ab, lane 2 is the immunoprecipitate with anti-GFP, and lane 3 is input.