Journal: American Journal of Physiology - Renal Physiology
Article Title: Effects of erythropoietin receptor activity on angiogenesis, tubular injury, and fibrosis in acute kidney injury: a “U-shaped” relationship
doi: 10.1152/ajprenal.00306.2017
Figure Lengend Snippet: Transient increase in renal erythropoietin receptor (EpoR) expression in ischemic-reperfusion injury (IRI)-induced acute kidney injury (AKI). AKI was induced in wild-type (WT) mice by IRI via renal arterial clamping for 30 min, followed by reperfusion. At predetermined time points, 6 mice from each group were killed and blood were collected for the measurement of plasma creatinine (PCr; A) and blood urea nitrogen (BUN; B). Statistical significance was assessed by unpaired Student’s t-test and significance was accepted when *P < 0.05, **P < 0.01, between 2 groups at same time point for A and B. C: mRNA of EpoR (left), EPO (middle), and neutrophil gelatinase-associated lipocalin (NGAL) (right) by quantitative polymerase chain reaction (qPCR). D: EpoR and β-common receptor (βCR) protein expression in the kidney. Left: representative immunoblots for EpoR determined by 3 EpoR antibodies (synthetic antibody Fab 6, commercial M-20, and monoclonal B9) and βCR. Right: summary of all immunoblots. Data are expressed as means ± SD (n = 6) from each group, statistical significance was evaluated by one-way ANOVA followed by Student-Newman-Keuls post hoc test, and significance was accepted when *P < 0.05, **P < 0.01, between 2 groups for C and D. E: characterization of new anti-EpoR monoclonal antibody. BaF3 cells, a murine interleukin 3 (IL-3)-dependent hematopoietic pro-B cell line, was purchased from American Type Culture Collection (Manassas, VA). BaF3-cell (EpoR-null) stably expressing Flag mouse EpoR cell line was generated by transfection of BaF3 cells with COOH-terminal Flag-tagged murine full-length EpoR retroviral vector. BaF3 cells transfected with empty vector only were used for negative control of EpoR expression. After confluence, cell lysate was prepared and subjected to immunoblot. Hybridoma supernatant from selected positive clones (by ELISA) was tested. Representative immunoblots for EpoR protein from 3 independent experiments using monoclonal anti-human EpoR antibodies (top), Flag-tagged EpoR protein with Flag antibody (middle), and β-actin protein (bottom) in native BaF3 cells and BaF3-expressing flag-tagged murine EpoR cells.
Article Snippet: Mouse monoclonal antibody against β-actin (β-actin) was from Sigma-Aldrich (St. Louis, MO); mouse monoclonal antibody against Akt, rabbit antibody against phospho-Akt, and rabbit antibody against cleaved caspase-3 were from Cell Signaling Technology (Danvers, MA); mouse monoclonal antibody against CD31 and rabbit polyclonal antibody against CD31 (CD31) were from Abcam (Cambridge, MA); goat polyclonal antibody against CD34 (CD34) was from R&D Systems; rabbit polyclonal antibody against human Ki67 (Ki67) and, mouse monoclonal antibody against connective tissue growth factor (CTGF) were from Abcam; mouse monoclonal antibody against common βCR was from R&D Systems; rabbit antibody against EpoR (EpoR, M-20) was from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit antibody against phospho- and total extracellular signal-regulated kinase (Erk) was from Cell Signaling Technology; mouse monoclonal antibody against hypoxia-inducible factor-1α (HIF1α) and rabbit polyclonal antibody against HIF2α were from Novus Biologicals (Littleton, CO); mouse antibody against phospho- c-Jun NH 2 -terminal kinase (JNK) and rabbit antibody against JNK were from Cell Signaling Technology; rabbit antibody against microtubule-associated light chain 3 (LC3) was from Novus Biologicals (Littleton, CO); guinea pig antibody against p62 (p62) was from Progen Biotechnik (Heidelberg, Germany); rabbit antibody against pimondazole with hydroxyprobe-1 kit [pimonidazole (PIM)] was from Pharmacia International (Belmont, MA); mouse monoclonal antibody against α-smooth muscle actin (α-SMA) was from Sigma-Aldrich (St. Louis, MO); rabbit monoclonal antibody against phospho-Stat5 was from Abcam; rabbit polyclonal antibody against VEGF-A (VEGF-A) for immunoblotting and goat polyclonal antibody against VEGF-A for immunohistochemistry were from Santa Cruz Biotechnology; and rat monoclonal antibody against flk-1/VEGFR2 (VEGFR2) was from BD PharMingen (San Jose, CA).
Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Stable Transfection, Generated, Transfection, Plasmid Preparation, Negative Control, Clone Assay, Enzyme-linked Immunosorbent Assay
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