Journal: Cardiovascular diabetology

Article Title: Klotho inhibits renal ox-LDL deposition via IGF-1R/RAC1/OLR1 signaling to ameliorate podocyte injury in diabetic kidney disease.

doi: 10.1186/s12933-023-02025-w

Figure Lengend Snippet: Fig. 6 Klotho regulated podocytic ox-LDL deposition via IGF-1R/RAC1/OLR1 signal axis. A IF analysis of the expression of Nephrin and RAC1 following administration of the inhibitor (PPP) and agonist (IGF2) of IGF-1R on KL+/− DKD and TgKL DKD, respectively. B Western blot analysis was conducted to determine the expression of RAC1, Podocin, and Cleaved Caspase3 following administration of the inhibitor (PPP) and agonist (IGF2) of IGF-1R on KL+/− DKD and TgKL DKD, respectively. C Representative western blot and summarized data showing the effects of administration of the inhibitor (PPP) and agonist (IGF2) of IGF-1R in the absence or presence of Klotho on the relative protein levels of OLR1, RAC1, Podocin, WT1 and Cleaved Caspase3. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: The antibodies used were listed below: anti-ox-LDL antibody (1:100, orb10973, Biorybt), anti-Podocin antibody (1:200, PA5-79757, Invitrogen), anti-Klotho antibody (1:100, PA5-88303, Invitrogen), anti-WT1 antibody (1:50, ab89901, Abcam), anti-PDGFRβ antibody (1:50, ab89901, Abcam), anti-GATA3 antibody (1:2400, ab199428, Abcam), anti-CXCL16 antibody (1:200, 60123- 1-Ig, Proteintech), anti-Parkin antibody (1:100, YT3593, ImmunoWay), anti-RAC1 antibody (1:20, AF385-SP, R&D), anti-Nephrin antibody (1:10, sc-376522, Santa Cruz), anti-OLR1 antibody (1:100, 11837-1-AP, Proteintech), anti-SYNPO antibody (1:200, 21064-AP, Proteintech), anti-IGF-1R antibody (1:10, sc-81464, Santa Cruz), goat polyclonal secondary antibody to mouse Alexa fluor 488 (1:400, ab150113, Abcam), goat polyclonal secondary antibody to rabbit Alexa fluor 555 (1:400, ab150078, Abcam), goat polyclonal secondary antibody to rabbit Alexa fluor 647 (1:400, ab150079, Abcam), goat antimouse Alexa fluor 568 (1:400, ab175473, Abcam), DAPI (1:1000, C1002, Beyotime).

Techniques: Expressing, Western Blot

Journal: Tissue Engineering Part A

Article Title: Induced Elastic Matrix Deposition Within Three-Dimensional Collagen Scaffolds

doi: 10.1089/ten.tea.2010.0749

Figure Lengend Snippet: FIG. 5. Effect of factors on enzyme synthesis and active forms. Representative immunoblots of (A) MMP-9 and (B) MMP-2 indicating the active and inactive protein bands for each case, and (C) LOX. (D) Representative gelatin zymo- gram of MMP-2 indicating the active enzyme band at 62 kDa and zymogen at 72 kDa. LOX, lysyl oxidase; MMP, matrix metalloproteinase.

Article Snippet: After this, the membranes were blocked in TBST (Tris-buffered saline with 0.01% v/v Tween-20, pH 7.4) containing 5% w/v milk for an hour, immunostained using polyclonal antibodies (1:1000 for 1.5 h) against LOX (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), MMP-2 (Abcam, Cambridge, MA), MMP-9 (Millipore), and HRPconjugated secondary antibody (1:10,000 for 1 h) (GE Healthcare Biosciences, Pittsburgh, PA), and detected using an ECL-plus chemiluminogen detection kit (GE Healthcare Biosciences).

Techniques: Western Blot

Journal: Diabetes

Article Title: High Glucose Increases Lysyl Oxidase Expression and Activity in Retinal Endothelial Cells: Mechanism for Compromised Extracellular Matrix Barrier Function

doi: 10.2337/db10-0365

Figure Lengend Snippet: A : Graph shows the effect of LOX siRNA on LOX protein level in RRECs. In cells grown in HG medium and transfected with LOX siRNA, the LOX protein expression was significantly decreased compared with that in cells grown in HG medium and transfected with scrambled siRNA. Data are expressed as mean ± SD (* P < 0.005, ** P < 0.05). B : Effect of reduced LOX activity on cell monolayer permeability and effect of reduced LOX expression on cell monolayer permeability. The permeability of FITC-conjugated dextran molecules was significantly decreased to near normal level in cells grown in HG medium after incubation with BAPN compared with RRECs grown in HG. Data are expressed as mean ± SD (* P < 0.05, n = 6; ** P < 0.05; n = 6). The permeability of FITC-conjugated dextran molecules was also significantly decreased to near normal level in cells grown in HG medium after transfection with LOX siRNA compared with untransfected RRECs grown in HG. Data are expressed as mean ± SD (*** P < 0.005; n = 3). N, normal.

Article Snippet: RRECs were transfected with LOX small interfering RNA (siRNA) (Santa Cruz, Santa Cruz, CA) and nonspecific siRNA (scrambled siRNA, 5′-aauauuggcguuaagauucua-3′, Ambion, Austin, TX) in the presence of 0.2% lipofectamin 2000 (Invitrogen) prepared in Opti-MEM (Invitrogen).

Techniques: Transfection, Expressing, Activity Assay, Permeability, Incubation