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7 protocols using anti lox

1

Modulation of Kidney Cell Signaling

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Normal rat kidney tubule epithelial cells (NRK‐52E cells), and HEK‐293 cells were cultured as described. Before the experiment, cells were starved overnight in a medium containing 0.5% FBS and then treated with drugs. AT1R‐β‐ARR biased agonist SII ([1‐sar, 4, 8‐ile]‐angiotensin II) was purchased from Jill biochemical and dissolved in ddH20 at a stock solution of 10 mM. Angiotensin II human was purchased from Sigma‐Aldrich (cat# 4474‐91‐3). PD98059 was purchased from Selleck (cat# 167869–21‐8).
The antibodies used for Western blot analysis included (a) anti‐ LOX (no. 31238), anti‐β‐arrestin1 (no. 32099), anti‐elastin (no. 21610), anti‐Col1 (no. 34710), and anti‐GAPDH (no. 82451), purchased from Abcam; (b) anti‐ ERK1/2 (no. 4695), and anti‐p‐ERK1/2 (no. 4370), purchased from Cell Signaling Technology; and (c) anti ‐STAT3 (sc‐482), anti‐STAT3‐pS727 (no. sc‐800R) and anti‐STAT3‐pY705 (sc‐7993) were purchased from Santa Cruz Biotechnology Inc. The secondary antibodies, including donkey anti‐rabbit IgG–horseradish peroxidase (sc‐2313), and goat anti‐mouse IgG–horseradish peroxidase (sc‐2005) were purchased from Santa Cruz Biotechnology Inc.
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2

Cell Signaling Modulators in ECM Interactions

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Gadolinium, anti N-cadherin neutralizing antibody, L-name, ML7, PMA and BAPN were from Sigma; anti integrin blocking antibody, Anti-collagen I, III, IV and VI, anti-TNC, anti-laminin, anti-biglycan, anti-decorin, anti-lumican, anti-LOX and anti-Pax9 antibodies were from Abcam; C3 and CN02 were from Cytoskeleton Inc; Y27632 and LY294002 were from Calbiochem; pertussis toxin, NSC23766, cytochalasin D, H89, C1, U0126, SB203580, SP600125, PP1, SB431542, SR11302 and mithramycin were from Tocris; and cytotoxic necrotizing factor 1 (CNF 1) was prepared as described previously (Mammoto et al., 2004 (link)).
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3

Immunofluorescence Labeling of Cellular Proteins

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Primary antibodies used were anti-FLAG (Sigma-Aldrich, F1804), anti-Myc and anti-HA (Medical & Biological Laboratories Co., M192-3S and M180-3, respectively), anti-LOX (Abcam, ab31238), anti-ATP7A (Abcam, ab13995), and anti–Golgin-97 (Cell Signaling Technology, 13192) antibodies. Secondary antibodies used were horseradish peroxidase–conjugated anti-mouse immunoglobulin G (IgG), anti-rabbit IgG, and anti-chicken IgY (Abcam, ab205719, ab6721, and ab6877, respectively) and Alexa Fluor 488–conjugated anti-mouse IgG and Alexa Fluor 555–conjugated anti-rabbit IgG (Thermo Fisher Scientific, A-11029 and A-31572, respectively).
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4

Immunocytochemistry Protocol for Quantifying Mitotic Lox Levels

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Forty-eight hours after transfection with siControl or siLOX, 250,000 cells were plated in glass coverslips in 6-well plates and allowed to attach for 24 hr at 37°C and in a 5% CO2 atmosphere. Cells were then fixed in 4% paraformaldehyde (PFA) for 15 min. After permeabilization with 70% ethanol, cells were blocked with 5% BSA in PBS-TT (0.5% Tween and 0.1% Triton) for 1 hr at RT. The cells were immunostained for LOX using monoclonal antibody anti-LOX (Abcam), anti-tubulin (Cell Signaling Technology or DSHB Univ. Iowa), and anti-pH3(Ser10) (Cell Signaling Technology). Then the cells were incubated with appropriate secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 569 for 1 hr (Life Technologies, Thermo Fisher Scientific). DNA was stained with DAPI (Vector Laboratories, Burlingame, CA). For the immunofluorescence staining of the mitotic fractions the samples were fixed in PFA 4%, washed and reconstituted in PBS before overnight incubation on poly-lysine coated cover slips (BD Biosciences) at 4°C. The samples were then stained with Hoechst 33342 (Sigma Aldrich) to detect DNA and with tubulin and LOX antibodies as described above. Fluorescence images were detected by confocal microscope NLO 710 Zeiss, and images were collected using Carl Zeiss Zen Software (Zeiss, Germany).
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5

Immunohistochemical Analysis of LOX and HIF-α

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After being deparaffinized and rehydrated, the tissue sections were incubated with anti-LOX (1:100 dilution, Abcam, Cambridge, CB, UK) or HIF-α antibodies overnight at 4 °C. Primary antibody binding to the tissues was detected using an HRP-conjugated secondary antibody. All the sections were stained with DAB and counterstained with hematoxylin. Images were taken in a 400 × field, quantified using IPP 6.0 software and assessed by two experimenters.
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6

Protein Extraction and Western Blot Analysis

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Total proteins were isolated by Total Extraction Kit (KGP2100; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) following the manufacturer's protocols. Protein concentration was determined by the BCA reagent kit (KGPBCA; Nanjing KeyGen Biotech Co., Ltd.). Equal amounts of protein (60 μg) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes, which were blocked in 5% non-fat milk, and then incubated with the primary antibodies against anti-HIF-1α (1:1,000; ProteinTech Group, Inc.), anti-TGF-β (1:500; Abcam, USA), anti-Smad4 (1:1,000; ProteinTech Group, Inc.), anti-LOX (1:2,000; Abcam, USA), anti-E-cadherin (1:200; Cell Signaling Technology, Danvers, MA, USA) and anti-β-actin (1:1,000; Elabscience Biotechnology Co., Ltd.). The samples were incubated with the antibodies overnight at 4°C. Subsequently, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1:6,000; OriGene, Beijing, China) at room temperature for 1 h. An enhanced chemiluminescence reagent (ECL kit, KGP1121; Nanjing KeyGen Biotech Co., Ltd.) was applied as a chromogenic substrate for 1 min, and then visualized with an Amersham Imager 600 instrument (GE Healthcare Life Sciences). Grayscale analysis was performed with ImageJ software.
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7

Immunohistochemistry and Immunoblotting of Aortic Lysates

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For immunohistochemistry, aortas from 6-wk-old C57BL/6 mice were embedded in cryoblock medium (VWR International) on dry ice. Cryosections were incubated with anti-LOX (Abcam, Cambridge, MA, USA) or anti-LOXL2 (Santa Cruz Biotechnology, Dallas, TX, USA) before fixation with acetone and further processing with secondary antibodies coupled to Alexa Fluor fluorochromes (Thermo Fisher Scientific) as previously described in Bignon et al. (9 (link)).
Frozen aortas were pulverized on dry ice before lysis in 25 mM Tris (pH 7.5), 100 mM NaCl, 5 mM EDTA, and 1% Triton X-100 containing Protease Inhibitor Cocktail (MilliporeSigma). Samples were centrifuged for 30 min at 15,000 g, and pellets were solubilized in Laemmli buffer. Proteins of both lysates were separated by SDS-PAGE and electrotransfered to PVDF membranes for immunodetection of LOX, LOXL2 (Abnova), fibronectin (Merck), and actin (Abcam).
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