anti-reg3γ  (Absolute Biotech Inc)

Journal: Gut microbes

Article Title: Intestinal epithelial pH-sensing receptor GPR65 maintains mucosal homeostasis via regulating antimicrobial defense and restrains gut inflammation in inflammatory bowel disease.

doi: 10.1080/19490976.2023.2257269

Figure Lengend Snippet: Figure 1. IEC-derived GPR65 preserves intestinal antimicrobial activity. (a-c) qPCR analysis of the indicated genes in IECs from the large bowels of Gpr65fl/fl and Gpr65ΔIEC mice. (d-f) qPCR analysis of the indicated genes in IECs from the small bowels of Gpr65fl/fl and Gpr65ΔIEC mice. (g) representative immunofluorescence staining of REG3γ in both large and small bowels from Gpr65fl/fl and Gpr65ΔIEC

Article Snippet: Primary antibodies against REG3γ (Cat: ab198216), β-actin (Cat: sc-8432), and GPR65 (Cat: PA5–111835) were purchased from Abcam (Cambridge, UK), Santa Cruz Biotechnology (Dallas, TX, USA), and Thermo Fisher Scientific, respectively.

Techniques: Derivative Assay, Activity Assay, Immunofluorescence, Staining

Journal: mSystems

Article Title: Paneth Cells Protect against Acute Pancreatitis via Modulating Gut Microbiota Dysbiosis

doi: 10.1128/msystems.01507-21

Figure Lengend Snippet: AP patients and experimental AP mice presented Paneth cell defects. (A) Histopathological changes and mean numbers of Paneth cells per crypt of duodenal mucosa specimens were assessed by H&E staining. Original magnification, ×200 ( n = 7 to 14 individuals per group). (B) Lysozyme expression (green) was assessed in Paneth cells of duodenal mucosa specimens by immunofluorescence (magnification, ×200) and lysozyme-positive/DAPI + quantification. (C to K) The mRNA expression levels of lysozyme (C), HD6 (D), HD5 (E), Reg3γ (F), Ang4 (G), sPLA2 (H), TGFβ (I), Wnt3a (J), and Lgr5 (K) were assessed. (L) Lysozyme expression (green) (magnification, ×200) and lysozyme-positive/DAPI + quantification in three AP models. (M) Mean number of Paneth cells per crypt in AP models. The data are presented as the means ± SD. ns, no significant difference; *, P ≤ 0.05.

Article Snippet: The membrane was blocked with 3% bovine serum albumin (BSA) for 1 h and incubated with primary antibodies, diluted in primary antibody dilution buffer (Epizyme Biotech, China), against Lgr5 (catalog number A10545; Abclonal, China), lysozyme (catalog number A0099; Dako, Denmark), Reg3γ (catalog number sc-377038; Santa Cruz Biotechnology, USA), Defa5 (catalog number A18208; Abclonal, China), Ang4 (catalog number sc-377497; Santa Cruz Biotechnology, USA), and sPLA2 (catalog number sc-58363; Santa Cruz Biotechnology, USA) overnight at 4°C.

Techniques: Staining, Expressing, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Gut REG3γ-Associated Lactobacillus Induces Anti-inflammatory Macrophages to Maintain Adipose Tissue Homeostasis

doi: 10.3389/fimmu.2017.01063

Figure Lengend Snippet: REG3γ overexpression causes alterations of gut microbiota and expands the pools of anti-inflammatory macrophages. (A) 16S rRNA analyses of gut microbiota in huREG3 γ tgIEC and control littermate WT mice. The weaned huREG3 γ tgIEC (REG3γ) and control littermate WT mice were reared in different cages, and then 16S rRNAs from pooled small intestinal content samples ( n = 5, 7- to 8-week-old male mice fed normal chow) were amplified using primers that targeted the V3–V4 regions of the 16S rRNA. The samples were clustered at genus levels using the sample genus count matrices. WT, wild-type; REG3γ, huREG3 γ tgIEC mice. See also NCBI with accession number SAMN04569211 and SAMN04569229. Data are a representative of three independent experiments. (B) Q-PCR of Lactobacillus genus in the ileum and proximal colon tissues of WT and huREG3 γ tgIEC mice fed normal chow ( n = 5). Standard curves were prepared from serial dilution of Escherichia coli genomic 16S rRNA. (C) Fluorescence in situ hybridization and immunostaining of ileum fragments in 7- to 8-week-old male huREG3 γ tgIEC mice (REG3γ) and control littermate WT mice. The representative images from five mice per group; Scale bars = 40 μm. (D) Flow cytometry of MHCII(+)F4/80(+), F4/80(+)CD11b(+), and F4/80(+)CD11C(+) macrophages in small intestinal (S.I) lamina propria of huREG3 γ tgIEC (REG3γ/REG3g) and control littermate WT mice. The proportion of MHCII(+)F4/80(+) macrophages in the S.I lamina propria of WT and huREG3 γ tgIEC mice were compared ( n = 6; lower). (E) Comparison of total MHCII(+)F4/80(+) macrophages in the whole S.I lamina propria of huREG3 γ tgIEC (REG3g) and control littermate WT mice ( n = 6). (F) Flow cytometry of F4/80(+)CD206(+), F4/80(+)Gr-1(+), and F4/80(+)IL-10(+) macrophages in the S.I lamina propria of huREG3 γ tgIEC and control littermate WT mice. The proportion of MHCII(+)F4/80(+), F4/80(+)Gr-1(+), and F4/80(+)IL-10(+) macrophages in WT and huREG3 γ tgIEC (REG3g) mice were compared ( n = 6). (G) Immunostaining of F4/80(+)CD206(+) macrophages in the ileum tissues of huREG3 γ tgIEC (REG3γ) and control littermate WT mice. The representative images from six mice per group; scale bars = 40 μm. * p < 0.05, ** p < 0.01, and *** p < 0.001 ( t -test, mean ± SD); NS, no significant. See also Figures S1–S3 in Supplementary Material.

Article Snippet: Anti- REG3γ (PA517, Thermo) and anti-MUC2 (H300, Santa) were also purchased.

Techniques: Over Expression, Control, Amplification, Serial Dilution, Fluorescence, In Situ Hybridization, Immunostaining, Flow Cytometry, Comparison

Journal: Frontiers in Immunology

Article Title: Gut REG3γ-Associated Lactobacillus Induces Anti-inflammatory Macrophages to Maintain Adipose Tissue Homeostasis

doi: 10.3389/fimmu.2017.01063

Figure Lengend Snippet: REG3γ-associated lactobacillus induces accumulation of macrophages in germ-free (GF) mice. (A) Flow cytometry of macrophages in small intestinal lamina propria (LP) of GF mice with or without L.NK1 or L.NK2 colonization. The proportion of MHCII(+)F4/80(+) macrophages in GF mice with (GF/L.NK1 or GF/L.NK2) and without (GF/NC) Lactobacillus colonization were compared (lower; n = 6). (B) Comparison of total MHCII(+)F4/80(+) macrophages in whole small intestinal LP of GF mice with (GF/L.NK.1 or GF/L.NK2) and without (GF/NC) Lactobacillus colonization ( n = 6). (C) Flow cytometry of MHCII(+)F4/80(+) macrophages in the PP and spleen of L.NK1 (GF/L.NK1) or L.NK2 (GF/L.NK2) colonized GF mice. The proportion of MHCII(+)F4/80(+) macrophages in GF mice with (GF/L.NK.1 or GF/L.NK2) and without (GF/NC) Lactobacillus colonization were compared (right; n = 6). (D) Comparison of total F4/80(+)IL-10(+) macrophages (upper) and interleukin (IL)-10 levels (lower) in the whole small intestinal LP of GF mice with (GF/L.NK.1 or GF/L.NK2) or without (GF/NC) Lactobacillus colonization ( n = 6). (E) Immunostaining of F4/80(+)IL-10(+) macrophages in the ileum tissues with or without L.NK.1 colonization. The representative images of six mice per group; scale bars = 40 μm. (F) Flow cytometry of F4/80(+)IL-10(+) macrophages in the spleen of L.NK.1 (GF/L.NK.1) or L.NK.2 (GF/L.NK2) colonized GF mice. The proportion of F4/80(+)IL-10(+) macrophages in GF mice with (GF/L.NK.1 or GF/L.NK2) and without (GF/NC) Lactobacillus colonization were compared (right; n = 6). Specific pathogen-free (SPF), wt mice raised in SPF environment; * p < 0.05, ** p < 0.01, and *** p < 0.001 (one-way analysis of variance, mean ± SD). See also Figures S4 and S5 in Supplementary Material.

Article Snippet: Anti- REG3γ (PA517, Thermo) and anti-MUC2 (H300, Santa) were also purchased.

Techniques: Flow Cytometry, Comparison, Immunostaining