Fig 1: Expressions of MrgprD in peritoneal macrophages and splenic T lymphocytes. a RT-PCR analyses to detect the expressions of Mrgprd in liver, DRG, aorta, primary smooth muscle cells (SMC), primary peritoneal macrophages (in triplicates) and splenic T lymphocytes (in triplicates). -RT was the negative control omitting the template. The liver was used as the control negative for Mrgprd expression and DRG and aorta were used as the positive controls. (b–b?) Double immunohistochemical stainings showing the expression of MrgprD protein (red, b and b?) in F4/80-positive peritoneal macrophages (green, b' and b?). (c–c?) Double immunohistochemical stainings showing the expression of MrgprD protein (red, c and c?) in CD3-positive T lymphocytes (green, c' and c?). The nuclei were counterstained with DAPI. Higher magnification views of a representative cell are shown as insets in each panel. Scale bars: 20 µm and 5 µm in inserts
Fig 2: The expressions of MrgprD in murine macrophage-like RAW 264.7 and human T lymphocyte Jurkat cell lines. (a, b) The expressions of MrgprD in RAW 264.7 and Jurkat cells were determined by RT-PCR (a) and Western blotting (b). The tissue lysate of liver was used as the negative control and the lysate of DRGs was the positive control. (c–c? and d–d?) Confocal images showed the presence of MrgprD protein (red) in RAW 264.7 (c–c?) and Jurkat cells (d–d?). Scale bars 20 µm. (e) The cytoplasmic/nuclear fractions were separately extracted from Raw 264.7 and Jurkat cells (in triplicates) and analyzed for the expressions of MrgprD, Lamin B and GAPDH by Western blotting. The expressions of MrgprD and GAPDH but not Lamin B, in cytoplasmic fractions were detected (upper panels). By contract, MrgprD was not expressed in the nuclear fractions of Raw 264.7 and Jurkat cells. The purity of nuclear fraction was determined by the expression of Lamin B and the absence of GAPDH (lower panels)
Fig 3: MrgprD is expressed in the resident macrophages and T lymphocytes in lamina propria of intestinal mucosa. a–f Double immunostaining using anti-MrgprD and anti-F4/80 showed the expression of MrgprD (red) in the F4/80-positive macrophages (green) in the lamina propria of ileum (a–c) and colon (d–f), respectively. g–l Double immunostaining using anti-MrgprD and anti-CD3 showed the expression of MrgprD (red) in the CD3-positive T lymphocytes (green) in the lamina propria of ileum (g–i) and colon (j–l), respectively. Scale bars 100 µm
Fig 4: MrgprD is expressed in intestinal smooth muscle cells. (a–a?) Immunostaining of MrgprD (red) in murine thoracic aorta. Bright-field image of cryostate section of aorta (a), the staining of anti-MrgprD in aorta (a') and the higher magnification view of a' showing distribution of MrgprD in smooth muscle layers of aorta (a?). (b–b?) Double immunostaining of mouse SM-MHC11 (green, b) and MrgprD (red, b') in the primary smooth muscle cells isolated from murine colon. The nuclei were counterstained with DAPI. Scale bars 100 µm
Fig 5: MrgprD expression pattern in murine intestinal tract determined by immunohistochemistry. (a–g) Immunostaining of MrgprD (red) in the different segments of intestinal tract from anterior to posterior, i.e., duodenum (a), jejunum (b), ileum (c), proximal colon (d), middle colon (e), distal colon (f) and rectum (g). (a'–g') The higher magnification images corresponding to (a–g) are shown, respectively. MrgprD IR was found in the smooth muscle layers (SML), muscularis mucosae (MM) and the lamina propria (LP) of the intestinal tract but not in the epithelium (Epi). (h) The negative control showing the absence of MrgprD IR in the ileum. The nuclei were counterstained with DAPI. Scale bars, 500 µm (in white) and 100 µm (in yellow)
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