Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: α-Klotho Expression in Human Tissues

doi: 10.1210/jc.2015-1800

Figure Lengend Snippet: α-Klotho isoforms, sequence, and Western blot. A, Structure of the two isoforms of α-Klotho. Isoform 1 represents the full-length protein and contains a signal sequence domain (SS), two homologous domains (KL1, KL2), a short transmembrane domain (TM), and a short cytoplasmic tail. Shown is the site of the epitope for the antibody used in our experiments: AA 800 to 900, KL2. This epitope is absent from Isoform 2, a soluble, secreted protein that arises from alternative RNA splicing and contains only AA 1–549, and where the terminal 15 residues are replaced by the sequence shown. B, The full-length α-Klotho protein sequence of 1012 AA is shown, with KL1 and KL2 shown in red and green respectively, and TM highlighted (black). The peptides giving rise to the PRM signature are also shown (bold typeface; common to isoforms 1 and 2, red; exclusive to full-length α-Klotho, isoform 1, blue). C and D, Western blot analysis of cell lysates (C) and tissues (D) supports the presence of the full-length α-Klotho. Full-length rh α-Klotho protein (rh-α-Klotho). EC, epithelial cells.

Article Snippet: Cell lysates were obtained from commercially available primary cell cultures (ScienCell Research Laboratories), including: Human Epidermal Keratinocyte Lysate-adult (product code HEKL-a, catalog no. 2116), Human Prostate Epithelial Cell Lysate (product code HPrEpiCL, catalog no. 4416), Human Mammary Epithelial Cell Lysate (product code HMEpiCL, catalog no. 7616), Human Renal Proximal Tubular Epithelial Cell Lysate (product code HRPTEpiCL, catalog no. 4106), and Human Neuron Lysate (product code HNL, catalog no. 1526).

Techniques: Sequencing, Western Blot

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: α-Klotho Expression in Human Tissues

doi: 10.1210/jc.2015-1800

Figure Lengend Snippet: α-Klotho protein expression and distribution in human epithelial and reproductive tissues. IHC, positive staining (brown) was found in all the cellular layers of the epidermis (A) and appendage tissue such as hair follicle and sebaceous gland (B). Intestinal expression was primarily found in epithelial cells as illustrated in jejunum (C) and colon (D). In reproductive tissues, positive staining was found in epithelial Sertoli cells (E), testosterone producing Leydig cells (illustrated with white arrows) of the testis (F), and epithelial cells of the prostate gland G). H–K, In mammary tissue (H), endometrium of uterus (I), and endometrium of salpinx (K), the epithelial cell layer was staining strongly for α-Klotho protein; insets are larger magnifications of the epithelial layer. n ≥ 5 for each tissue.

Article Snippet: Cell lysates were obtained from commercially available primary cell cultures (ScienCell Research Laboratories), including: Human Epidermal Keratinocyte Lysate-adult (product code HEKL-a, catalog no. 2116), Human Prostate Epithelial Cell Lysate (product code HPrEpiCL, catalog no. 4416), Human Mammary Epithelial Cell Lysate (product code HMEpiCL, catalog no. 7616), Human Renal Proximal Tubular Epithelial Cell Lysate (product code HRPTEpiCL, catalog no. 4106), and Human Neuron Lysate (product code HNL, catalog no. 1526).

Techniques: Expressing, Staining

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: α-Klotho Expression in Human Tissues

doi: 10.1210/jc.2015-1800

Figure Lengend Snippet: Mass spectrometry characterization of the transmembrane α-Klotho protein in human tissues and cells: extracellular α-Klotho peptide GLFYVDFLSQKD (exon 3). A–D, Representative mass spectrometry spectra (left) and Skyline data (right) confirmed the presence of full-length α-Klotho rh full-length α-Klotho protein (rh-α-Klotho) (A), renal proximal tubular epithelial cells (B), kidney tissue (C), and renal artery (D). E–G, The full-length specific (isoform 1) αKlotho peptide LWITMNEPYTR (exon 4). Representative mass spectrometry spectra (left) and Skyline data (right) confirmed the presence of full-length α-Klotho. E), rh full-length α-Klotho protein (rh-α-Klotho); F, kidney tissue; G, renal artery; and H, neuronal cells.

Article Snippet: Cell lysates were obtained from commercially available primary cell cultures (ScienCell Research Laboratories), including: Human Epidermal Keratinocyte Lysate-adult (product code HEKL-a, catalog no. 2116), Human Prostate Epithelial Cell Lysate (product code HPrEpiCL, catalog no. 4416), Human Mammary Epithelial Cell Lysate (product code HMEpiCL, catalog no. 7616), Human Renal Proximal Tubular Epithelial Cell Lysate (product code HRPTEpiCL, catalog no. 4106), and Human Neuron Lysate (product code HNL, catalog no. 1526).

Techniques: Mass Spectrometry, Targeted Proteomics

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: α-Klotho Expression in Human Tissues

doi: 10.1210/jc.2015-1800

Figure Lengend Snippet: Confirmation of Transmembrane α-Klotho Protein Expression in Human Tissues and Cells

Article Snippet: Cell lysates were obtained from commercially available primary cell cultures (ScienCell Research Laboratories), including: Human Epidermal Keratinocyte Lysate-adult (product code HEKL-a, catalog no. 2116), Human Prostate Epithelial Cell Lysate (product code HPrEpiCL, catalog no. 4416), Human Mammary Epithelial Cell Lysate (product code HMEpiCL, catalog no. 7616), Human Renal Proximal Tubular Epithelial Cell Lysate (product code HRPTEpiCL, catalog no. 4106), and Human Neuron Lysate (product code HNL, catalog no. 1526).

Techniques: Expressing, Recombinant

Journal: PLoS ONE

Article Title: Is prnt a Pseudogene? Identification of Ram Prt in Testis and Ejaculated Spermatozoa

doi: 10.1371/journal.pone.0042957

Figure Lengend Snippet: APPA labeling of ram germinal cells in seminiferous tubules (E). Positive cells were detected (arrows) in spermatogonia (C), spermatocytes (D), spermatides (A), and spermatozoa (B). Negative control using mouse control serum (pre-immune), as the primary antibody (F). Same results were obtained when incubating sections with the secondary antibody alone (data not shown). Vimentin (G) and Ki-67 antigen (H), immunodetection of Sertoli cells cytoskeletal and spermatides, respectively. Spermatogonia (Sg), spermatocytes (Sc), spermatides (Sp), spermatozoa (Spz), Sertoli cell (S). Magnifications (A, B, C and D) ×1000, (E and F) ×100.

Article Snippet: To precisely determine the different cellular types present at the seminiferous tubules, and thus facilitate the identification of the cells expressing Prt, two other antibodies were used: one directed against Vimentin (Monoclonal Mouse Anti-Vimentin, clone Vim 3B4, Code M7020, Dako, Dilution 1/200), and the other against Ki-67 (Monoclonal Mouse Anti-Human Ki-67 antigen, clone MIB-1, Code M7240, Dako, Dilution 1/100), to locate respectively the Sertoli cell cytoskeleton, and the germinal cells with particular emphasis to the spermatides, as Ki-67 is a nuclear protein that is mainly expressed in proliferating cells .

Techniques: Labeling, Negative Control, Immunodetection