- PA5-85267 - Invitrogen Antibodies ARG1 antibody

Submitted references HA-coated collagen nanofibers for urethral regeneration via in situ polarization of M2 macrophages.

Niu Y, Stadler FJ, Yang X, Deng F, Liu G, Xia H
Journal of nanobiotechnology 2021 Sep 22;19(1):283

DA-DRD5 signaling controls colitis by regulating colonic M1/M2 macrophage polarization.

Liu L, Wu Y, Wang B, Jiang Y, Lin L, Li X, Yang S
Cell death & disease 2021 May 17;12(6):500

Clusterin regulates macrophage expansion, polarization and phagocytic activity in response to inflammation in the kidneys.

Weng X, Zhao H, Guan Q, Shi G, Feng S, Gleave ME, Nguan CC, Du C
Immunology and cell biology 2021 Mar;99(3):274-287

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Arginase 1 in various whole cell extracts using Arginase 1 polyclonal antibody (Product # PA5-85267) using 30 µg of sample at a dilution of 1:1000. Prior to incubation with primary antibody, the sample was separated on 12% SDS-PAGE.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Arginase 1 in rat tissue extract using Arginase 1 polyclonal antibody (Product # PA5-85267) using 50 µg of sample at a dilution of 1:10000. Sample was then incubated with HRP-conjugated anti-rabbit IgG secondary antibody. Prior to incubation with primary antibody, the sample was separated on 12% SDS-PAGE.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Arginase 1 in non-transfected (–) and transfected (+) HepG2 whole cell extracts using Arginase 1 polyclonal antibody (Product # PA5-85267) using 30 µg of sample at a dilution of 1:1000. Sample was then incubated with HRP-conjugated anti-rabbit IgG secondary antibody. Prior to incubation with primary antibody, the sample was separated on 10% SDS-PAGE.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Arginase 1 was performed by separating 50 µg of mouse tissue extract by 12% SDS-PAGE. Proteins were transferred to a membrane and probed with a Arginase 1 Polyclonal Antibody (Product # PA5-85267) at a dilution of 1:10000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of Arginase 1 was performed by separating 30 µg of various whole cell extracts by 12% SDS-PAGE. Proteins were transferred to a membrane and probed with a Arginase 1 Polyclonal Antibody (Product # PA5-85267) at a dilution of 1:1000 and a HRP-conjugated anti-rabbit IgG secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of Arginase 1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRIPSR760199_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of Arginase 1 was performed by loading 30 µg of Hep G2 Cas9 (Lane 1) and Hep G2 Arginase 1 KO (Lane 2) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-Arginase 1 Polyclonal Antibody (Product # PA5-85267, 1:2000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:5000 dilution) using the iBright™ FL 1500 (Product # A44115). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to Arginase 1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-Arginase 1 Polyclonal Antibody (Product # PA5-85267) and a 35kDa band corresponding to Arginase-1 was observed.. Tissue extracts (20 µg lysate) of Mouse Kidney (Lane 1), Rat Kidney (Lane 2), Mouse Liver (Lane 3) and Rat Liver (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:2000) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).Band of interest was observed only in Liver tissue and not in Kidney.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry analysis of Arginase 1 in 4% paraformaldehyde-fixed HeLa cells using Arginase 1 polyclonal antibody (Product # PA5-85267) at a dilution of 1:200.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry (Frozen) analysis of Arginase-1 was performed in frozen-sectioned mouse hippocampus tissue using Arginase 1 Polyclonal Antibody (Product # PA5-85267) at a dilution of 1:250 (Green). Blue: Fluoroshield with DAPI.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry (Paraffin) analysis of Arginase 1 was performed in paraffin-embedded mouse liver tissue using Arginase 1 Polyclonal Antibody (Product # PA5-85267) at a dilution of 1:500. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of Arginase 1 in HepG2 cells (control: unlabeled cells) using Arginase 1 polyclonal antibody (Product # PA5-85267) at a dilution of 1:50. Dylight 488-conjugated was used as a secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 CLU expression is associated with decreased ratio of NOS 2 + (M1 marker)/ARG 1 + (M2 marker) cells at day 7 following renal IRI. ARG 1 + and NOS 2 + cells were identified by immunohistochemistry as described in the ""Methods"" section. (a) The numbers of ARG 1 + cells. * P = 0.0010 (WT versus KO, two-tailed t -test). (b) The numbers of NOS 2 + cells. ** P = 0.0021 (WT versus KO, two-tailed t -test). (c) The difference in the M1/M2 ratio between the WT and CLU-KO groups was confirmed by the NOS 2 + /ARG 1 + ratio in each kidney. *** P = 0.0179 (WT versus KO, two-tailed t -test). Data in a , b and c are representative of three independent experiments and presented as the mean +- s.d. ( n = 3). (d) Representative images of three independent experiments. ARG 1 + (top 2) and NOS 2 + (bottom 2) cells (dark brown) in the tubulointerstitial area, the perivascular space and injured tubular epithelium of the renal cortical and medullary junction. Red arrowheads represent positively stained cells; Black arrowheads correspond to false-positive staining. Scale bar: 100 um. ARG 1; arginase 1; CLU, clusterin; DT, distal convoluted tubule; G, glomerulus; IA, interlobular artery; KO, clusterin knockout; NOS 2, nitric oxide synthase 2; PT, proximal convoluted tubule; WT, wild type.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation analysis of Arginase 1 in HepG2 whole cell extracts with Arginase 1 polyclonal antibody (Product # PA5-85267) using 5 µg of sample, followed by anti-Rabbit IgG secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 5 The deficiency of DRD5 signaling increased colonic M1 macrophages but reduced M2 cells in the colon of DSS colitis mice. A Immunofluorescent labeling of F4/80 + , Arg1 + , and Inos + colonic macrophages of WT and DRD5 -/- mice on day 6 after DSS treatment (top). The cell numbers are quantified by Image pro plus (bottom) ( n = 3). Scale bars, 200 mum. B Flow cytometry analysis of colonic macrophages of WT and DRD5 -/- mice ( n = 5 mice per group) on day 6 after DSS treatment as indicated. Data are presented as representative plots (top) and summary graphs of quantified percentages (bottom). C Flow cytometry analysis of the CD45.2 + cells percentage in Arg1 + and CD86 + colonic macrophages of lethally irradiated mice reconstituted with DRD5 -/- CD45.2/WT CD45.1 bone marrow (1:1 ratio), and a control WT CD45.2/WT CD45.1 group (1:1 ratio) on day 6 after DSS treatment ( n = 3 mice per group). Data are presented as representative plots (left) and summary graphs of quantified percentages (right). Data are pooled from two or three independent experiments. Error bars show means +- SEM. * p < 0.05, **p < 0.01, *** p < 0.001. Two-tailed unpaired student's t -test ( A , B , C ).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 CLU expression is associated with decreased ratio of NOS 2 + (M1 marker)/ARG 1 + (M2 marker) cells at day 7 following renal IRI. ARG 1 + and NOS 2 + cells were identified by immunohistochemistry as described in the ""Methods"" section. (a) The numbers of ARG 1 + cells. * P = 0.0010 (WT versus KO, two-tailed t -test). (b) The numbers of NOS 2 + cells. ** P = 0.0021 (WT versus KO, two-tailed t -test). (c) The difference in the M1/M2 ratio between the WT and CLU-KO groups was confirmed by the NOS 2 + /ARG 1 + ratio in each kidney. *** P = 0.0179 (WT versus KO, two-tailed t -test). Data in a , b and c are representative of three independent experiments and presented as the mean +- s.d. ( n = 3). (d) Representative images of three independent experiments. ARG 1 + (top 2) and NOS 2 + (bottom 2) cells (dark brown) in the tubulointerstitial area, the perivascular space and injured tubular epithelium of the renal cortical and medullary junction. Red arrowheads represent positively stained cells; Black arrowheads correspond to false-positive staining. Scale bar: 100 um. ARG 1; arginase 1; CLU, clusterin; DT, distal convoluted tubule; G, glomerulus; IA, interlobular artery; KO, clusterin knockout; NOS 2, nitric oxide synthase 2; PT, proximal convoluted tubule; WT, wild type.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 4 The polarization of macrophages to M2 phenotype is related to the elongated cell shape. A Fluorescence micrographs of Raw 264.7 macrophages immune-stained for arginase-1 (green), iNOS (red), and nuclear counterstain (blue) on cell plate (control), collagen and HA-collagen nanofibrous films. Scale bars: 15 mum. B Representative Western blot of arginase-1, iNOS, and tubulin of control, collagen and HA-collagen nanofibrous films and quantification of average across three separate experiments. Quantified TNF-alpha C and IL-10 D secretion from macrophages cultured on different nanofibrous scaffolds or culture plates using ELISA assay. n = 3, ** p < 0.01, *** p < 0.001

PA5-85267

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