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: Cells

Article Title: Role of Aberrantly Activated Lysophosphatidic Acid Receptor 1 Signaling Mediated Inflammation in Renal Aging

doi: 10.3390/cells10102580

Figure Lengend Snippet: SA-β-gal stain and double immunofluorescence stain in senescence-induced cells: ( A ) representative image of SA-β-gal staining in senescence-induced HRPTEpiC; original magnification, ×200. ( B ) The HRPTEpiC treated with 100 nM DOXO and 100μM H 2 O 2 showed increases in the number of SA-β-gal-positive cells. ( C ) Representative image of double immunofluorescence staining of LPAR1 and NF-κB in senescent HRPTEpiC; original magnification, ×200. ( D ) LPAR1- and ( E ) NF-κB-positive area increased in senescence-induced HRPTEpiC. ‡ p < 0.001.

Article Snippet: Human renal proximal tubular epithelial cells (HRPTEpiC) (ScienCell, Carlsbad, CA, USA) were grown in an epithelial cell medium (ScienCell, Carlsbad, CA, USA) containing epithelial cell growth supplement (ScienCell, Carlsbad, CA, USA), in a humidified atmosphere of 95% air and 5% CO 2 at 37 °C.

Techniques: Staining, Immunofluorescence, Double Immunofluorescence Staining

Journal: Cells

Article Title: Role of Aberrantly Activated Lysophosphatidic Acid Receptor 1 Signaling Mediated Inflammation in Renal Aging

doi: 10.3390/cells10102580

Figure Lengend Snippet: The ATX, LPAR1, PI3K, NF-κB, and inflammatory cytokines expression in senescent HRPTEpiC: ( A – C ) Representative Western blots demonstrating ATX, LPAR1, PI3K, NF-κB, and inflammatory cytokines expression in senescent cells. ( D , E ) LPAR1 and ( F , G ) NF-κB expression increased with DOXO and H 2 O 2 treatment. ( H – L ) The ATX, PI3K, and inflammatory cytokines expression up-regulated in senescence-induced HRPTEpiC. * p < 0.05, † p < 0.01, ‡ p < 0.001.

Article Snippet: Human renal proximal tubular epithelial cells (HRPTEpiC) (ScienCell, Carlsbad, CA, USA) were grown in an epithelial cell medium (ScienCell, Carlsbad, CA, USA) containing epithelial cell growth supplement (ScienCell, Carlsbad, CA, USA), in a humidified atmosphere of 95% air and 5% CO 2 at 37 °C.

Techniques: Expressing, Western Blot

Journal: Cells

Article Title: Role of Aberrantly Activated Lysophosphatidic Acid Receptor 1 Signaling Mediated Inflammation in Renal Aging

doi: 10.3390/cells10102580

Figure Lengend Snippet: The expression of LPAR1, PI3K, NF-κB, and inflammatory cytokines in senescence-induced HRPTEpiC before and after si- LPAR1 and si- N F -κB transfection: ( A ) representative Western blots of each protein involved. ( B – G ) LPAR1, PI3K, NF-κB, and inflammatory cytokine expression highly increased with senescence-induction of DOXO and H 2 O 2 treatment, while si- LPAR1 treatment reduced the increases. (a: si-c vs. si-L, si-N; b: si-c+D vs. si-L+D, si-N+H; c: si-c+H vs. si-L+H, si-N+H; si-c = si-control, si-L = si- LPAR1 , si-N = si- NF- κ B , D = doxorubicin, H = H 2 O 2 ). ‡ p < 0.001.

Article Snippet: Human renal proximal tubular epithelial cells (HRPTEpiC) (ScienCell, Carlsbad, CA, USA) were grown in an epithelial cell medium (ScienCell, Carlsbad, CA, USA) containing epithelial cell growth supplement (ScienCell, Carlsbad, CA, USA), in a humidified atmosphere of 95% air and 5% CO 2 at 37 °C.

Techniques: Expressing, Transfection, Western Blot, Control

Journal: Nephrology Dialysis Transplantation

Article Title: CRIM1 is localized to the podocyte filtration slit diaphragm of the adult human kidney

doi: 10.1093/ndt/gfn743

Figure Lengend Snippet: Results from real-time quantitative RT-PCR assessment of CRIM1 mRNA levels in two separate cultures of conditionally immortalized podocytes (Podocytes 1 and 2), normal human kidney samples (Normal 1 and 2), primary human renal proximal tubule cells (RPTEC 1 and 2) and primary human pulmonary artery smooth muscle cells (HPASMC 1 and 2). Results are in arbitrary units ± SD. The levels were normalized to three separate housekeeping genes and the identity of the PCR product was confirmed by sequencing.

Article Snippet: Primary human renal proximal tubular cells (RPTEC) were obtained from Cambrex Bio Science (Walkersville MD, USA).

Techniques: Quantitative RT-PCR, Sequencing