Characterization of NT629 antibody. (A) Site of single nucleotide deletions in MYH9 exon 40 and amino acid sequence of NMMHC-IIA carboxyl-terminal region. Nucleotides encoding amino acids are numbered beginning with initiation codon. Deduced amino acid sequence is written under nucleotide sequence. Deletions of a single guanine nucleotide (bold type) results in frameshift that cause replacement by aberrant amino acids at the carboxyl terminus (bold type) and premature termination. Epitopes of PRB440P and NT629 antibodies are underlined. (B) Total proteins of 293T cells transiently transfected with vectors expressing N-terminal myc-tagged wild-type MYH9 rod (1278-1960 aa) and 5818delG mutant MYH9 rod (1278-1946 aa) were analyzed by immunoblotting. Band corresponding to recombinant myc-tagged NMMHC-IIA rod was detected with antimyc antibody in both wild-type MYH9– and 5818delG MYH9–-transfected cells. PRB440P and NT629 detected recombinant NMMHC-IIA rod only in wild-type MYH9–transfected cells and in 5818delG MYH9–transfected cells (arrow head), respectively. Endogenous 220-kDa NMMHC-IIA band was detected in 293T cells with PRB440P. (C) Immunofluorescence analysis of 293T cells transiently transfected with N-terminal myc-tagged wild-type MYH9 rod and 5818delG MYH9 rod expression vectors double stained with PRB440P and antimyc mouse antibody, and with NT629 and antimyc rabbit antibody, respectively. PRB440P and NT629 intensely stained cytoplasm only in wild-type– and only in 5818delG-transfected cells, respectively. PRB440P weakly stained endogenous NMMHC-IIA in nontransduced 293T cells. (D) Immunofluorescence analysis of peripheral blood smears from a patient with E1841K double stained with PRB440P and NT629. PRB440P showed abnormal type I NMMHC-IIA localization in all neutrophils with diffusely stained background, and diffuse cytoplasmic staining in lymphocytes, monocytes, and platelets. In contrast, cells were not stained with NT629 antibody. (E) Immunofluorescence analysis of peripheral blood smears from a mosaic patient for 5818delG double-stained with PRB440P and NT629. PRB440P showed abnormal type I NMMHC-IIA localization in approximately 10% neutrophils, in which inclusion bodies were stained with NT629. Normal neutrophils with diffuse NMMHC-IIA distribution were not stained with NT629. (F) Immunoblots of buffy coat samples from healthy control and from patients 1 (5770delG) and 2 (5818delG). NT629 detected band corresponding to NMMHC-IIA polypeptide and additional small band in blood from patients, but not control.