polyclonal goat anti mouse areg antibody  (R&D Systems)

Journal: The FASEB Journal

Article Title: FOXP3⁺ Macrophage‐Derived Amphiregulin Promotes White Matter Repair of Experimental Autoimmune Encephalomyelitis

doi: 10.1096/fj.202500075rr

Figure Lengend Snippet: FIGURE 4 | AREG secreted by FOXP3+ macrophages promotes remyelination of OPCs. (A) Venn diagram on ligands produced by macrophages for OPCs differentiation among NicheNet, genes exhibiting FOXP3 binding as determined by CUT&RUN, and the differentially upregulated genes in FOXP3+ BMDM from the bulk RNA-seq. (B) Representative sections labeled with IBA1 (red) and AREG (green) of WT EAE mice and CKO EAE mice at the chronic phase were displayed. N = 5, compared with the WT control group, by one-way ANOVA (mean ± SD). Scale bar = 20 μm. (C) EAE peak phase spinal cord slices were displayed with IBA1 (green), AREG (red) and FOXP3 (blue). Chi-square and Fisher exact test were calculated for correlation between FOXP3 expression and AREG expression in macrophages. (D) Expression of AREG in FOXP3+ macrophages by flow cytometry. N = 5, compared with FOXP3− cells, by Student's t test (mean ± standard deviation). (E) Culture supernatants of WT and Foxp3-KO BMDM (± myelin pre-treated) were collected and assessed AREG concentration with ELISA. Experiments repeated 3 times, compared with WT BMDM, by one-way ANOVA (mean ± SD). (F) Expression of Areg in WT and Foxp3-KO BMDM (± myelin treated) by qPCR. Experiments repeated 3 times, by Student's t test (mean ± SD). (G) IGV visual analysis of the FOXP3 binding peak in the Areg promoter region from CUT&RUN. The arrows refer to the poten- tial binding site of FOXP3. (H) Quantitative analysis using anti-FOXP3 antibody and qPCR. Experiments were repeated 3 times, by Student's t test (mean ± SD). **p < 0.01, ***p < 0.001.

Article Snippet: The following primary antibodies were used: rabbit anti- Olig2 (Abcam, ab109186, 1:300), mouse anti- CC1 (Abcam, ab16794, 1:1000), rabbit anti- MBP (Proteintech, 10458- 1- AP, 1:500), rabbit anti- NG2 (Abcam, ab129051, 1:500), rabbit anti- dMBP (Millipore, ab5864, 1:2000), chicken antiNeurofilament H (Biolegend, 822601, 1:400), mouse anti- Ki67 (Abcam, ab279653, 1:500), mouse polyclonal anti- FOXP3 (Invitrogen, 14- 5773, 1:500), rabbit monoclonal anti- FOXP3 (Invitrogen, 700914, 1:500), rabbit anti- LC3A/B (Cell signaling technology, 12 741T, 1:1000), rabbit anti- Iba1 (Wako, 019- 19741, 1:1000), goat anti- AREG (R&D Systems, AF989, 1:500), and rabbit polyclonal anti- TMEM119 (catalog PA5- 119617, Invitrogen, 1:500).

Techniques: Produced, Binding Assay, RNA Sequencing, Labeling, Control, Expressing, Flow Cytometry, Standard Deviation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: An amphiregulin reporter mouse enables transcriptional and clonal expansion analysis of reparative lung Treg cells

doi: 10.1101/2024.09.26.615245

Figure Lengend Snippet: A) Left: schematic depicting genetic targeting of the endogenous Areg locus via homologous recombination with the P2A- Thy1.1 -STOP-Neomycin knock-in construct, with subsequent crossing to the FLPR mouse to remove the neomycin cassette and create the final Areg Thy1.1 allele. Right: A depiction of Areg transcription/translation in the Areg Thy1.1 mouse; for each molecule of Areg mRNA translated, a single Thy1.1 mRNA is also translated (as a separate protein, due to the P2A site) and traffics separately to the surface of the cell, where it can be targeted by fluorescently conjugated antibodies. B) Mouse splenocytes from Areg Thy1.1 mice underwent a short-term stimulation protocol (PMA/ionomycin, 3h), then were stained for AREG using traditional methods (i.e., intracellularly, with biotinylated polyclonal antibody staining followed by streptavidin-mediated amplification) or for Thy1.1 (live staining with a fluorophore-conjugated antibody). Percentages of total Treg cells are shown in quadrants on plot. Representative staining shown from 2 experiments. C) Schematics depicting the models of lung damage used in this study, including general timecourse and disease characteristics, and Treg cell increases/Areg production status. D) Thy1.1 staining by flow cytometry on lung Tregs from Areg Thy1.1 mice during the IAV or bleomycin (bleo.) models of lung damage, or from control (saline-treated) lungs. Days post-instillation (dpi) for each model indicated in figure. Representative staining from 2-3 experiments. See Fig. S2A for gating strategy throughout figure.

Article Snippet: Following surface marker staining, for staining with anti-mouse Foxp3-FITC (clone FJK-16s; Invitrogen), anti-mouse Ki67-AlexaFluor700 (clone SolA15; Invitrogen), anti-mouse α-smooth muscle actin-eFluor660 (clone 1A4; Invitrogen), or biotinylated anti-mouse AREG (endogenous) (polyclonal goat IgG; R&D systems) followed by Streptavidin conjugates, cells were fixed/permeabilized with Foxp3/Transcription Factor Staining Buffer Kit (Cytek).

Techniques: Homologous Recombination, Knock-In, Construct, Staining, Amplification, Flow Cytometry, Control, Saline