Mouse OCILRP2/CLEC2i Antibody

Detection of Mouse OCILRP2/CLEC2i by Flow Cytometry

Membrane re-localization of OCILRP2 via treatment with an anti-CD3/CD28 mAb.EL4 cells were incubated with plate-bound CD3/CD28 antibodies, isotype-matched mIgGs, or PMA/ionomycin for 48 h. The cells (1×106 cells) were then incubated with an anti-OCILRP2 antagonist antibody for 30 min on ice, followed by staining with an FITC-labeled second antibody. The cells were analyzed with Cellquest software using a FACS Calibur flow cytometer. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0113218), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of OCILRP2/CLEC2i by Western Blot

Downregulation of Ocilrp2 transcription in macrophages upon innate stimuli. (A) qRT-PCR analysis of Ocilrp2 mRNA levels in mouse peritoneal macrophages following LPS, Poly (I:C) or CpG stimulation or VSV, and SeV infection for 4 h. Data were normalized to the expression of GAPDH. (B) qRT-PCR analysis of Ocilrp2 mRNA levels after LPS stimulation on 2, 4, 8, and 12 h in mouse peritoneal macrophages. Untreated macrophages were used as a control. Data were normalized to the expression of GAPDH. (C) Western Blot analysis of Ocilrp2 protein levels after stimulated by LPS in mouse peritoneal macrophages. Ocilrp2 expression was analyzed by western blotting. Western blots and immunofluorescence images are representative of three independent experiments. (D, E) qRT-PCR analysis of Ocilrp2 mRNA level in macrophages treated with NF-kappa B inhibitor (BAY11-7082) or Erk inhibitor (SCH772984) for 1 h and then stimulated with LPS for 4 h or infected with VSV for 4 h. Data were normalized to the expression of GAPDH. Data were calculated from at least 3 independent experiments (means ± s.e.m.), **P< 0.01 (The two-tailed Student’s t-test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36300111), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of OCILRP2/CLEC2i by Western Blot

Silencing of Ocilrp2 promotes IL-6 expression in vitro and in vivo. (A) Peritoneal macrophages were transfected with scrambled siRNA (siCtrl) or three Ocilrp2 siRNAs (siOcilrp2-1, siOcilrp2-2, siOcilrp2-3) for 48 h, Ocilrp2 mRNA levels were detected with qRT-PCR, and Ocilrp2 protein levels were analyzed by Western Blot. The experiment was repeated three times. (B) qRT-PCR analysis of Ocilrp2 mRNA level in mouse peritoneal macrophages transfected with Ocilrp2 siRNA and 48 h later stimulated with LPS or infected with VSV for the indicated time.(C) qRT-PCR analysis of IL-6 mRNA level in macrophages transfected as in (A), and 48 h later stimulated with LPS for the indicated time and (D) ELISA of IL-6 in supernatants of macrophages stimulated with LPS for 8 h. (E) qRT-PCR analysis of IL-6 mRNA level in macrophages transfected as in (A), and 48 h later after VSV infection for the indicated time. (F) ELISA of IL-6 in supernatants of macrophages transfected as in (A), and 48 h later infection with VSV for 8 h. (G, H) Six C57BL/6 mice were transfected in vivo with Ocilrp2 siRNA or Control siRNA (2.5 mg/kg) for 2 days, respectively. Then, peritoneal macrophages and serum were collected at 12 h after intraperitoneal injection of LPS (10 mg/kg). qRT-PCR analysis of Ocilrp2 and IL-6 mRNA level in mouse peritoneal macrophages and (I) ELISA of IL-6 in serum. Data were calculated from at least 3 independent experiments (means ± s.e.m.), *P< 0.05, **P< 0.01 (The two-tailed Student’s t-test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36300111), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of OCILRP2/CLEC2i by Western Blot

Silencing of Ocilrp2 promotes IL-6 expression in vitro and in vivo. (A) Peritoneal macrophages were transfected with scrambled siRNA (siCtrl) or three Ocilrp2 siRNAs (siOcilrp2-1, siOcilrp2-2, siOcilrp2-3) for 48 h, Ocilrp2 mRNA levels were detected with qRT-PCR, and Ocilrp2 protein levels were analyzed by Western Blot. The experiment was repeated three times. (B) qRT-PCR analysis of Ocilrp2 mRNA level in mouse peritoneal macrophages transfected with Ocilrp2 siRNA and 48 h later stimulated with LPS or infected with VSV for the indicated time.(C) qRT-PCR analysis of IL-6 mRNA level in macrophages transfected as in (A), and 48 h later stimulated with LPS for the indicated time and (D) ELISA of IL-6 in supernatants of macrophages stimulated with LPS for 8 h. (E) qRT-PCR analysis of IL-6 mRNA level in macrophages transfected as in (A), and 48 h later after VSV infection for the indicated time. (F) ELISA of IL-6 in supernatants of macrophages transfected as in (A), and 48 h later infection with VSV for 8 h. (G, H) Six C57BL/6 mice were transfected in vivo with Ocilrp2 siRNA or Control siRNA (2.5 mg/kg) for 2 days, respectively. Then, peritoneal macrophages and serum were collected at 12 h after intraperitoneal injection of LPS (10 mg/kg). qRT-PCR analysis of Ocilrp2 and IL-6 mRNA level in mouse peritoneal macrophages and (I) ELISA of IL-6 in serum. Data were calculated from at least 3 independent experiments (means ± s.e.m.), *P< 0.05, **P< 0.01 (The two-tailed Student’s t-test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36300111), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of OCILRP2/CLEC2i by Western Blot

Silencing of Ocilrp2 accelerates the activation of the TLR4 signaling. Mouse peritoneal macrophages were treated with or without LPS (1 µg/ml) for different periods, as indicated, after transfection with siCtrl or siOcilrp2-3. (A)Then subjected to Western blot analyses using anti-Myd88, anti-Traf6, and anti- phosphorylated (p-) or total proteins of IKK alpha / beta, I kappa B alpha, and p65. (B) Densitometric quantification of the ratio of Myd88, Traf6, p-IKK alpha / beta, p-I kappa B alpha and p-p65. Data were analyzed by three independent experiments. (C) Immunoblot analysis of phosphorylated (p-) or total proteins of pErk, Jnk, and p38 for the indicated time. The beta -actin was detected as a loading control. (D) Densitometric quantification of the ratio of p-Erk, p-Jnk and p-p38. Data were analyzed by three independent experiments. (E) Mouse peritoneal macrophages were transfected with Ocilrp2 siRNA and 48hr later stimulated with LPS for the indicated times. Immunofluorescence analysis of the nuclear translocation of p65 and the co-localization between p65 (green) and cell nucleus (blue) (Bar, 50 μm). (F) Quantifying of p65 protein translocation based on the visual images in c, ~300 cells for each time point. (G) Immunoblot analysis of p65 protein in cytoplasm and nucleus of mouse peritoneal macrophages transfected with Ocilrp2 siRNA and 48 h later stimulated by LPS for the indicated time. Lamin A/C is used as an internal nuclear control. GAPDH is shown as a cytoplasm internal control. Data represent of the results of three independent experiments (means ± s.e.m). Significant differences compared to the control group are denoted by *P < 0.05, **P < 0.01 (two-tailed Student’s t-test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36300111), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of OCILRP2/CLEC2i by Western Blot

Silencing of Ocilrp2 accelerates the activation of the TLR4 signaling. Mouse peritoneal macrophages were treated with or without LPS (1 µg/ml) for different periods, as indicated, after transfection with siCtrl or siOcilrp2-3. (A)Then subjected to Western blot analyses using anti-Myd88, anti-Traf6, and anti- phosphorylated (p-) or total proteins of IKK alpha / beta, I kappa B alpha, and p65. (B) Densitometric quantification of the ratio of Myd88, Traf6, p-IKK alpha / beta, p-I kappa B alpha and p-p65. Data were analyzed by three independent experiments. (C) Immunoblot analysis of phosphorylated (p-) or total proteins of pErk, Jnk, and p38 for the indicated time. The beta -actin was detected as a loading control. (D) Densitometric quantification of the ratio of p-Erk, p-Jnk and p-p38. Data were analyzed by three independent experiments. (E) Mouse peritoneal macrophages were transfected with Ocilrp2 siRNA and 48hr later stimulated with LPS for the indicated times. Immunofluorescence analysis of the nuclear translocation of p65 and the co-localization between p65 (green) and cell nucleus (blue) (Bar, 50 μm). (F) Quantifying of p65 protein translocation based on the visual images in c, ~300 cells for each time point. (G) Immunoblot analysis of p65 protein in cytoplasm and nucleus of mouse peritoneal macrophages transfected with Ocilrp2 siRNA and 48 h later stimulated by LPS for the indicated time. Lamin A/C is used as an internal nuclear control. GAPDH is shown as a cytoplasm internal control. Data represent of the results of three independent experiments (means ± s.e.m). Significant differences compared to the control group are denoted by *P < 0.05, **P < 0.01 (two-tailed Student’s t-test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36300111), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of OCILRP2/CLEC2i by Western Blot

Downregulation of Ocilrp2 transcription in macrophages upon innate stimuli. (A) qRT-PCR analysis of Ocilrp2 mRNA levels in mouse peritoneal macrophages following LPS, Poly (I:C) or CpG stimulation or VSV, and SeV infection for 4 h. Data were normalized to the expression of GAPDH. (B) qRT-PCR analysis of Ocilrp2 mRNA levels after LPS stimulation on 2, 4, 8, and 12 h in mouse peritoneal macrophages. Untreated macrophages were used as a control. Data were normalized to the expression of GAPDH. (C) Western Blot analysis of Ocilrp2 protein levels after stimulated by LPS in mouse peritoneal macrophages. Ocilrp2 expression was analyzed by western blotting. Western blots and immunofluorescence images are representative of three independent experiments. (D, E) qRT-PCR analysis of Ocilrp2 mRNA level in macrophages treated with NF-kappa B inhibitor (BAY11-7082) or Erk inhibitor (SCH772984) for 1 h and then stimulated with LPS for 4 h or infected with VSV for 4 h. Data were normalized to the expression of GAPDH. Data were calculated from at least 3 independent experiments (means ± s.e.m.), **P< 0.01 (The two-tailed Student’s t-test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36300111), licensed under a CC-BY license. Not internally tested by R&D Systems.