Characterization of the Human Hair Keratin–Associated Protein 2 (KRTAP2) Gene Family Hiroki Fujikawa, Atsushi Fujimoto, Muhammad Farooq, Masaaki Ito,

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Characterization of the Human Hair Keratin–Associated Protein 2 (KRTAP2) Gene Family Hiroki Fujikawa, Atsushi Fujimoto, Muhammad Farooq, Masaaki Ito, Yutaka Shimomura Journal of Investigative Dermatology Volume 132, Issue 7, Pages 1806-1813 (July 2012) DOI: 10.1038/jid.2012.73 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Identification of size polymorphisms in the human hair keratin–associated protein (KRTAP)2 genes. (a) Physical map of the human KRTAP2 genes on chromosome 17q12-21. Direction of the genes is indicated by the black arrows. (b) PCR amplification of the KRTAP2-2 (upper panel) and the KRTAP2-4 (lower panel) genes in nine Japanese (J1–J9) and nine Caucasian (C1–C9) individuals. (c) Schematic representation of the KRTAP2-2, KRTAP2-4 genes, and their size polymorphisms. The 15-bp (base pair) insertion into the open reading frame (ORF) of the KRTAP2-2v1 and the KRTAP2-2v2 alleles is indicated by the green boxes and black arrows. Insertion into the 3′-untranslated region (3′-UTR) is indicated by the yellow boxes. Asterisks and black dots indicate the sequence identity. MWM, molecular weight markers. Journal of Investigative Dermatology 2012 132, 1806-1813DOI: (10.1038/jid.2012.73) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Characterization of human hair keratin–associated protein (KRTAP)2-1 protein. (a) Co-immunoprecipitation (co-IP) assays between the hemagglutinin (HA)-KRTAP2-1 and Flag-KRTAP2-1. After IP was performed with anti-HA agarose beads, western blots (WBs) were performed using either anti-Flag or anti-HA antibodies. (b) Results of glutathione S-transferase (GST) pull-down assays. GST-KRTAP2-1 and HA-KRTAP2-1 proteins were overexpressed in competent cells and human embryonic kidney (HEK)293T cells, respectively. Subsequently, these proteins were then used for the pull-down assays with Glutathione Sepharose beads. (c) WBs with the anti-KRTAP2 antibody. The antibody specifically recognized the HA-KRTAP2-1 protein, which was overexpressed in HEK293T cells, as well as the endogenous KRTAP2 proteins in human hairs. As there were 17 additional amino acid residues at the N terminus of the HA-KRTAP2-1 protein, this led to a larger product as compared with the native KRTAP2 proteins. (d) Indirect immunofluorescence analysis of the human hair follicle section using anti-KRTAP2 antibody. KRTAP2 is predominantly expressed in the keratinizing zone of the human hair shaft cortex. Counterstaining with 4′,6-diamidino-2-phenylindole is shown in blue. Bar=100μm. Journal of Investigative Dermatology 2012 132, 1806-1813DOI: (10.1038/jid.2012.73) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Hair keratin–associated protein (KRTAP)2-1 protein specifically binds to hair keratins in vitro. (a) Co-immunoprecipitation (co-IP) between Flag-KRTAP2-1 and K85. (b) Co-IP between hemagglutinin (HA)-KRTAP2-1 and Flag-K34, as well as between HA-KRTAP2-1 and Flag-K86. The cell lysate was precipitated with anti-Flag agarose beads (a, b). (c) Co-IP between HA-KRTAP2-1 and Flag-K10, as well as between HA-KRTAP2-1 and Flag-K86. (d) Co-IP between HA-KRTAP2-1 and K71. (e) Co-IP between HA-KRTAP2-1 and endogenously expressed β-actin. The cell lysate was precipitated with anti-HA agarose beads (c–e). Note that KRTAP2-1 showed affinity for the hair keratins (K85 (a), K34 (b), and K86 (b, c)). By contrast, it did not bind to the epithelial keratins (K10 (c) and K71 (d)) or β-actin (e). WB, western blot. Journal of Investigative Dermatology 2012 132, 1806-1813DOI: (10.1038/jid.2012.73) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Involvement of the head domain of K86 in the binding to hair keratin–associated protein (KRTAP)2-1. (a) Co-immunoprecipitation (co-IP) between hemagglutinin (HA)-KRTAP2-1 and Flag-tagged truncated K86 proteins. The cell lysate was precipitated with anti-Flag agarose beads. Among the three truncated K86 proteins, only the one with the head domain (Flag-K86-H&R (head and rod)) was co-precipitated with HA-KRTAP2-1. Full-length K86 (Flag-K86-F) was used as a positive control. Amino-acid residues for each domain of K86 are shown on the top panel. (b) Results of glutathione S-transferase (GST) pull-down assays. GST fusion proteins for truncated K86 and HA-KRTAP2-1 proteins were overexpressed in competent cells and human embryonic kidney (HEK)293T cells, respectively. These were then used for the pull-down assays with the Glutathione Sepharose beads. Only the GST fusion protein for the head domain of K86 (GST-K86-H) showed affinity to HA-KRTAP2-1. WB, western blot. Journal of Investigative Dermatology 2012 132, 1806-1813DOI: (10.1038/jid.2012.73) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions