Anti-LOXL2 antibody

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-LOXL2 antibody (AB96233)

Immunofluorescence analysis of 4% paraformaldehyde fixed at RT for 15 mins HeLa cells labelling LOXL2 protein at nucleus using ab96233 at a 1/500 dilution (Green). Alpha tubulin, a cytoskeleton marker stained by anti-alpha tubulin antibody at a 1/1000 dilution (Red).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-LOXL2 antibody (AB96233)

Immunocytochemistry/ Immunofluorescence analysis of A431 cells labeling LOXL2 with ab96233 at 1 : 500 (green). Cells were fixed in ice cold methanol for 5 minutes. Cells were co-stained with Hoechst 33342 (blue).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LOXL2 antibody (AB96233)

Immunohistochemical analysis of Paraffin-embedded human esophageal carcinoma tissue labeling LOXL2 with ab96233 at 1/3000 dilution. Antigen Retrieval : Citrate buffer, pH 6.0, 15 min (Cuisinart Electric Pressure Cooker #EPC-1200, choose "high pressure"). Endogenous peroxidase blocking : 3% H₂O₂, RT, 30min. Blocking : 1.5% goat serum (dilute goat serum by 1xPBS), RT, 30 min. Primary antibody incubation : 4°C overnight. Secondary antibody incubation : ABC HRP Kit (Rabbit IgG), 1 : 200, RT, 30min. Washing condition : PBS, 2 x 5 mins. Chromogen system : DAB.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LOXL2 antibody (AB96233)

IHC image of ab96233 staining in human testis formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab96233, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• I-ELISA

Supplier Data

Indirect ELISA - Anti-LOXL2 antibody (AB96233)

Indirect ELISA analysis was performed by coating the plate with recombinant Human LOXL2 protein, His tagged (44.94-0.7 nM). Coated protein was probed with ab96233 (1 μg/mL). A Goat anti-rabbit IgG antibody (HRP) conjugated (1 : 10000) was used to detect the bound primary antibody.

• IHC

CiteAb

Immunohistochemistry - Anti-LOXL2 antibody (AB96233)

Immunohistochemistry using Anti-LOXL2 antibody, ab96233. Publication image from Chen, P. S. et al., 2016, Nat Commun, 27966531. Legend direct from paper.

LOXL2 correlates with collagen crosslinking and diastolic abnormalities in HFpEF.(a,b) Immunostaining of LOXL2 (a) and COL1A (b) in heart tissues of control subjects and HfpEF patients. Scale bars, 100 µm. Blue : haematoxylin. Brown : LOXL2 (a) or COL1A (b). (c–e) Correlation of crosslinked collagen with echocardiographic E/E′ ratio (c), left ventricular end-diastolic pressure (LVEDP, d) and LOXL2 (e). r : Pearson coefficient. (f–h) Correlation of LOXL2 level with COL1A (f), echocardiographic E/E′ ratio (g) and left ventricular end-diastolic pressure (LVEDP, h) in human studies. r : Pearson coefficient.

• WB

CiteAb

Western blot - Anti-LOXL2 antibody (AB96233)

Western Blotting using Anti-LOXL2 antibody, ab96233. Publication image from Chen, P. S. et al., 2016, Nat Commun, 27966531. Legend direct from paper.

LOXL2 acts with TGF-β through PI3K/ATK/mTORC1 signalling to control myofibroblast transformation and migration.(a) Western blot analysis of LOXL2, SMAD2, p-SMAD2 and GAPDH in human primary cardiac fibroblasts transfected with control (Ctrl) or LOXL2 siRNA. (b) Quantification of TGF-β isoforms in the culture media of human primary cardiac fibroblasts transfected with control or LOXL2 siRNA (n=4). P value : Student's t-test. Error bar : s.e.m. (c) Changes of COLA1,α-SMA and FN1 mRNA in human primary cardiac fibroblasts transfected with control (Ctrl) or LOXL2 siRNA. (d) Western blot of LOXL2, p-AKT, AKT, p-S6K, S6K and p-4E-BP1 with or without PI3K inhibitors LY294002/PI828 (10 µM each), PI3Kα inhibitors A66/BYL719 and mTORC1 inhibitor Rapamycine (0.1 µM) in cells infected with Ad_GFP/Ad_LOXL2. (e) TGF-β2 protein in the culture media (n=4). P value : Student's t-test. Error bar : s.e.m. (f,g) Western blot (f) and quantification (g) of TGF-β2 protein in the mice heart ventricles 6 weeks after sham/TAC operation. n=4 mice per group. P value : Student's t-test. Error bar : s.e.m. (h) A signalling cascade from LOXL2 to PI3K/AKT/mTORC1 to TGF-β2 translation. (i) Representative phase-contrast image of human primary cardiac fibroblasts 72 h after transfection with control (Ctrl) or LOXL2 siRNA. Scale bars, 50 µm. (j) Immunostaining of Col1A in IgG1- orα-LOXL2-treated mice 10 weeks (10W) after sham or TAC operation. Scale bars, 100 µm. Blue : haematoxylin. Brown : Col1A. (k,l) Gap closure assay of fibroblast migration in control, TGF-β2,α-LOXL2 (k) groups and quantification of cells migrating into the gap (l). Scale bars, 200 µm. n=5–10, P value : Student's t-test. Error bar : s.e.m.

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• WB

CiteAb

Western blot - Anti-LOXL2 antibody (AB96233)

Western Blotting using Anti-LOXL2 antibody, ab96233. Publication image from Chen, P. S. et al., 2016, Nat Commun, 27966531. Legend direct from paper.

LOXL2 acts with TGF-β through PI3K/ATK/mTORC1 signalling to control myofibroblast transformation and migration.(a) Western blot analysis of LOXL2, SMAD2, p-SMAD2 and GAPDH in human primary cardiac fibroblasts transfected with control (Ctrl) or LOXL2 siRNA. (b) Quantification of TGF-β isoforms in the culture media of human primary cardiac fibroblasts transfected with control or LOXL2 siRNA (n=4). P value : Student's t-test. Error bar : s.e.m. (c) Changes of COLA1,α-SMA and FN1 mRNA in human primary cardiac fibroblasts transfected with control (Ctrl) or LOXL2 siRNA. (d) Western blot of LOXL2, p-AKT, AKT, p-S6K, S6K and p-4E-BP1 with or without PI3K inhibitors LY294002/PI828 (10 µM each), PI3Kα inhibitors A66/BYL719 and mTORC1 inhibitor Rapamycine (0.1 µM) in cells infected with Ad_GFP/Ad_LOXL2. (e) TGF-β2 protein in the culture media (n=4). P value : Student's t-test. Error bar : s.e.m. (f,g) Western blot (f) and quantification (g) of TGF-β2 protein in the mice heart ventricles 6 weeks after sham/TAC operation. n=4 mice per group. P value : Student's t-test. Error bar : s.e.m. (h) A signalling cascade from LOXL2 to PI3K/AKT/mTORC1 to TGF-β2 translation. (i) Representative phase-contrast image of human primary cardiac fibroblasts 72 h after transfection with control (Ctrl) or LOXL2 siRNA. Scale bars, 50 µm. (j) Immunostaining of Col1A in IgG1- orα-LOXL2-treated mice 10 weeks (10W) after sham or TAC operation. Scale bars, 100 µm. Blue : haematoxylin. Brown : Col1A. (k,l) Gap closure assay of fibroblast migration in control, TGF-β2,α-LOXL2 (k) groups and quantification of cells migrating into the gap (l). Scale bars, 200 µm. n=5–10, P value : Student's t-test. Error bar : s.e.m.

false

• WB

CiteAb

Western blot - Anti-LOXL2 antibody (AB96233)

Western Blotting using Anti-LOXL2 antibody, ab96233. Publication image from Chen, P. S. et al., 2016, Nat Commun, 27966531. Legend direct from paper.

LOXL2 correlates with cardiac dysfunction in both mouse model and human patients.(a) Quantification of Loxl2 mRNA expression in the heart 1–10 weeks after sham/TAC operation. n=4–5 mice per group. P value : Student's t-test. Error bar : standard error of the mean (s.e.m.). (b) Western blot analysis of Loxl2 protein in the mice heart ventricles 1–7 weeks after sham/TAC operation. P value : Student's t-test. Error bar : s.e.m. (c) Representative immunostaining of Loxl2 in the heart 1, 2 and 7 weeks after sham/TAC operation. Scale bars, 100 µm. Blue : haematoxylin. Brown : Loxl2. (d) Representative immunostaining of Col1A andα-SMA in the heart 1 week after sham/TAC operation. Scale bars, 100 µm. Blue : haematoxylin. Brown : Col1A/α-SMA. (e–g) Determination of total, soluble and insoluble collagen content of left ventricles from sham- or TAC-operated mice 1, 2 and 10 weeks after the procedure (n=5 in each group). The amounts of total and soluble collagen were determined by measuring hydroxyproline content of total and pepsin-acid solubilized left ventricles, respectively. The amount of insoluble collagen was calculated by subtracting the amount of soluble collagen from total collagen. (h) Correlation of left ventricular fractional shortening (LVFS) with the insoluble collagen content of left ventricles after sham or TAC operation (n=5 per group). r : Pearson coefficient. (i–k) mRNA quantification of LOX genes in ventricular tissues of healthy human donor hearts (n=8; i) or from patients with ischaemic (n=8; j) or idiopathic dilated (n=10; k) cardiomyopathy. IDCM, idiopathic dilated cardiomyopathy. P value : Student's t-test. Error bar : s.e.m. (l) Immunostaining of LOXL2 (brown) in human heart tissues obtained from control subjects and patients with ischaemic (ISCH) or idiopathic dilated cardiomyopathy (IDCM). Scale bars, 20 µm. Blue : haematoxylin. Brown : Loxl2. (m,n) Correlation of the mRNA level of LOXL2 with COL1A (m) and COL3A (n) in the ventricular tissues of patients with idiopathic dilated cardiomyopathy (IDCM). r : Pearson coefficient. Black circles : cardiomyopathy tissues (n=10). Open circles : control heart tissues (n=8).

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