Journal: Experimental and molecular pathology

Article Title: Expression of free fatty acid receptor 2 in normal and neoplastic tissues.

doi: 10.1016/j.yexmp.2024.104902

Figure Lengend Snippet: Fig. 1. Specificity analysis of the rabbit polyclonal anti-human free fatty acid receptor 2 (FFAR2) antibody 0524 by Western blot analyses. (A-E) Western blot analysis of membrane preparations of BON-1 cells that endogenously express FFAR2 or (F) from normal human liver. (B) To establish controls for adsorption, the anti-FFAR2 antibody 0524 was preincubated for 2 h with 10 μg/ml of immunising peptide (+ peptide). (C) Western blot analysis of membrane preparations of BON-1 cells after transfection with siRNA targeting FFAR2. To evaluate the potential sensitivity of the antibody to possible phosphorylation of the serine or threonine residues contained in the C-terminus of the receptor, (D) the samples were either treated with lambda phosphatase before Western blot analysis or (E) the purification steps were carried out without phosphatase inhibitors in the buffer. Ladder indicates migration of protein molecular weight markers (kDa). All results are representative of three independent experiments.

Article Snippet: For double-labelling fluorescence immunohistochemistry, sections were incubated overnight at 4 ◦C with rabbit anti-FFAR2 0524 antibody (1:100 dilution) together with mouse monoclonal anti-insulin antibody (1:100 dilution; Abcam), mouse monoclonal anti-glucagon antibody (1:500 dilution; Sigma-Aldrich, St. Louis, MO, USA), rat monoclonal anti-somatostatin-14/28 antibody (1:300 dilution; Abcam, Cambridge, UK), guinea pig polyclonal anti-gastrin antibody (1:800 dilution; Origene, Rockville, MD, USA), rat monoclonal anti-ghrelin antibody (1:50; R&D Systems/biotechne, Wiesbaden, Germany), mouse monoclonal anti-serotonin antibody (1:50 dilution; DAKO, Glostrup, Denmark), mouse monoclonal anti-gastric inhibitory polypeptide (GIP) antibody (1:1500 dilution; Invitrogen, Waltham, MA, USA), mouse monoclonal anti-glucagon-like peptide-1 (GLP-1) antibody (1:500 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), or mouse monoclonal antipeptide YY antibody (1:100 dilution; Novus Biologicals/biotechne, Wiesbaden, Germany).

Techniques: Western Blot, Membrane, Adsorption, Transfection, Phospho-proteomics, Purification, Migration, Molecular Weight

Journal: Experimental and molecular pathology

Article Title: Expression of free fatty acid receptor 2 in normal and neoplastic tissues.

doi: 10.1016/j.yexmp.2024.104902

Figure Lengend Snippet: Fig. 2. Specificity analysis of the rabbit polyclonal anti-human free fatty acid receptor 2 (FFAR2) antibody 0524 by immunocytochemistry. (A) BON-1 or (G) OH-1 cells that endogenously express FFAR2 as well as (H) HEK-293 cells that do not express the FFAR2 were fixed and stained with the anti-FFAR2 antibody 0524, followed by an Alexa Fluor 488- or Cy3-conjugated anti-rabbit secondary antibody. To analyse antibody specificity, BON-1 cells were treated with (B) scrambled siRNA or (C) FFAR2-specific siRNA. (D) To establish adsorption controls, the anti-FFAR2 antibody was preincubated for 2 h with 10 μg/ml of immunising peptide. (E) To evaluate the potential sensitivity of the antibody to possible phosphorylation of serine or threonine residues contained in the C-terminus of the receptor, the samples were preincubated with lambda phosphatase before immunocytochemistry analysis. (F) To demonstrate agonist-induced internalisation of the receptor, BON-1 cells were treated with 1.75 nM acetic acid for 30 min. (I) As a further control, HEK-293 cells were transiently transfected with plasmids encoding for the human FFAR2. Green colour (A-G) or red colour (H, I) represents FFAR2; blue colour represents 4′,6-diamidino-2-phenylindole (DAPI)-stained DNA. Scale bar, 100 μm (A–F). All results are representative of three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: For double-labelling fluorescence immunohistochemistry, sections were incubated overnight at 4 ◦C with rabbit anti-FFAR2 0524 antibody (1:100 dilution) together with mouse monoclonal anti-insulin antibody (1:100 dilution; Abcam), mouse monoclonal anti-glucagon antibody (1:500 dilution; Sigma-Aldrich, St. Louis, MO, USA), rat monoclonal anti-somatostatin-14/28 antibody (1:300 dilution; Abcam, Cambridge, UK), guinea pig polyclonal anti-gastrin antibody (1:800 dilution; Origene, Rockville, MD, USA), rat monoclonal anti-ghrelin antibody (1:50; R&D Systems/biotechne, Wiesbaden, Germany), mouse monoclonal anti-serotonin antibody (1:50 dilution; DAKO, Glostrup, Denmark), mouse monoclonal anti-gastric inhibitory polypeptide (GIP) antibody (1:1500 dilution; Invitrogen, Waltham, MA, USA), mouse monoclonal anti-glucagon-like peptide-1 (GLP-1) antibody (1:500 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), or mouse monoclonal antipeptide YY antibody (1:100 dilution; Novus Biologicals/biotechne, Wiesbaden, Germany).

Techniques: Immunocytochemistry, Staining, Adsorption, Phospho-proteomics, Control, Transfection

Journal: Frontiers in Immunology

Article Title: A novel necroptosis-related gene index for predicting prognosis and a cold tumor immune microenvironment in stomach adenocarcinoma

doi: 10.3389/fimmu.2022.968165

Figure Lengend Snippet: Individual assessment of NRGPI genes and association with necroptosis. (A) Spearman’s correlation between NRGPI genes and infiltration of macrophages in the TCGA STAD dataset assessed with TIMER database. (B) Spearman’s correlation between NRGPI genes and KEGG TGF-β signaling pathway signature genes and (C) WNT signaling pathway signature genes. (D) mRNA expression level of RIPK1, RIPK3, MLKL (necroptosis core mediators), NRGPI oncogenes (SERPINE1, GPX3, GRP, FCN1, CYTL1, CNTN1, PLCL1, and APOD), and markers of WNT signaling pathway (WNT2B, WNT9A) and TGF- signaling pathway (TGFB1, TGFB3) in gastric cancer cells (AGS and MNK45) after being treated with a combination of human recombinant TNF-α, SMAC mimetic, and zVAD-FMK (TSZ) to induce necroptosis or added necroptosis inhibitor (necrostatin-1) to inhibit necroptosis. Graph shows mean ± SD. *P<0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not signficant.

Article Snippet: After washing with 10% phosphate buffered saline (PBS), the sections were blocked with 5% BSA and incubated with primary antibodies against RIPK1 (Proteintech, #17519-1-AP, Rabbit, 1:50), RIPK3 (Proteintech, #17563-1-AP, Rabbit, 1:100), MLKL (Proteintech, #21066-1-AP, Rabbit, 1:50), SERPINE1 (Proteintech, #13801-1-AP, Rabbit, 1:50), FCN1 (Proteintech, #11775-1-AP, Rabbit, 1:50), CNTN1 (Proteintech, #13843-1-AP, Rabbit, 1:50), CYTL1 (Proteintech, #15856-1-AP, Rabbit, 1:50), PLCL1 (Abcam, #EPR11213, Rabbit, 1:100), GRP (Proteintech, #28482-1-AP, Rabbit, 1:500), GPX3 (Affinity Biosciences, #DF6765, Rabbit, 1:50), APOD (Proteintech, #10520-1-AP, Rabbit, 1:50), TGFB1 (Affinity Biosciences, #AF1027, Rabbit, 1:100), TGFB3 (Proteintech, #18942-1-AP, Rabbit, 1:50), WNT2B (Affinity Biosciences, #DF12538, Rabbit, 1:100), WNT9A (Affinity Biosciences, #DF9044, Rabbit, 1:100), CD68 (Abcam,ab955,1:3000), CD206 (Cell Signaling,#24595,1:200), and CD163 (Cell Signaling,#93498,1:250) at 4°C overnight.

Techniques: Expressing, Recombinant

Journal: Frontiers in Immunology

Article Title: A novel necroptosis-related gene index for predicting prognosis and a cold tumor immune microenvironment in stomach adenocarcinoma

doi: 10.3389/fimmu.2022.968165

Figure Lengend Snippet: Representative images of expression (brown, cell cytoplasmic/nucleus stain) of RIPK1, RIPK3, MLKL (necroptosis core mediators), NRGPI oncogenes (SERPINE1, GPX3, GRP, FCN1, CYTL1, CNTN1, PLCL1, and APOD), markers of WNT signaling pathway (WNT2B, WNT9A), TGF-β signaling pathway (TGFB1, TGFB3), and macrophage (CD63, CD206, CD163) in the clinical samples of stomach adenocarcinoma.

Article Snippet: After washing with 10% phosphate buffered saline (PBS), the sections were blocked with 5% BSA and incubated with primary antibodies against RIPK1 (Proteintech, #17519-1-AP, Rabbit, 1:50), RIPK3 (Proteintech, #17563-1-AP, Rabbit, 1:100), MLKL (Proteintech, #21066-1-AP, Rabbit, 1:50), SERPINE1 (Proteintech, #13801-1-AP, Rabbit, 1:50), FCN1 (Proteintech, #11775-1-AP, Rabbit, 1:50), CNTN1 (Proteintech, #13843-1-AP, Rabbit, 1:50), CYTL1 (Proteintech, #15856-1-AP, Rabbit, 1:50), PLCL1 (Abcam, #EPR11213, Rabbit, 1:100), GRP (Proteintech, #28482-1-AP, Rabbit, 1:500), GPX3 (Affinity Biosciences, #DF6765, Rabbit, 1:50), APOD (Proteintech, #10520-1-AP, Rabbit, 1:50), TGFB1 (Affinity Biosciences, #AF1027, Rabbit, 1:100), TGFB3 (Proteintech, #18942-1-AP, Rabbit, 1:50), WNT2B (Affinity Biosciences, #DF12538, Rabbit, 1:100), WNT9A (Affinity Biosciences, #DF9044, Rabbit, 1:100), CD68 (Abcam,ab955,1:3000), CD206 (Cell Signaling,#24595,1:200), and CD163 (Cell Signaling,#93498,1:250) at 4°C overnight.

Techniques: Expressing, Staining

Journal: Frontiers in Immunology

Article Title: A novel necroptosis-related gene index for predicting prognosis and a cold tumor immune microenvironment in stomach adenocarcinoma

doi: 10.3389/fimmu.2022.968165

Figure Lengend Snippet: (A) Expression level (IHC quantification) of RIPK1, RIPK3, MLKL (necroptosis core mediators), NRGPI oncogenes (SERPINE1, GPX3, GRP, FCN1, CYTL1, CNTN1, PLCL1, and APOD), and markers of WNT signaling pathway (WNT2B, WNT9A), TGF-β signaling pathway (TGFB1, TGFB3), and macrophage (CD63, CD206, CD163) in the clinical samples (n = 4) of stomach adenocarcinoma. The scattered dots represent the IHC score of each individual sample. The thick middle lines represent the median value and error bars indicate the standard deviation. The bottom and top of the boxes are the 25th and 75th percentiles (interquartile range), respectively. (B) A correlation matrix illustrating Pearson’s correlation coefficients indicating the relationship among expression levels of RIPK1, RIPK3, MLKL (necroptosis core mediators), NRGPI oncogenes (SERPINE1, GPX3, GRP, FCN1, CYTL1, CNTN1, PLCL1, and APOD), and markers of WNT signaling pathway (WNT2B, WNT9A), TGF-β signaling pathway (TGFB1, TGFB3), and macrophage (CD63, CD206, CD163) in the clinical samples (n = 4) of stomach adenocarcinoma. P values are shown as: *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: After washing with 10% phosphate buffered saline (PBS), the sections were blocked with 5% BSA and incubated with primary antibodies against RIPK1 (Proteintech, #17519-1-AP, Rabbit, 1:50), RIPK3 (Proteintech, #17563-1-AP, Rabbit, 1:100), MLKL (Proteintech, #21066-1-AP, Rabbit, 1:50), SERPINE1 (Proteintech, #13801-1-AP, Rabbit, 1:50), FCN1 (Proteintech, #11775-1-AP, Rabbit, 1:50), CNTN1 (Proteintech, #13843-1-AP, Rabbit, 1:50), CYTL1 (Proteintech, #15856-1-AP, Rabbit, 1:50), PLCL1 (Abcam, #EPR11213, Rabbit, 1:100), GRP (Proteintech, #28482-1-AP, Rabbit, 1:500), GPX3 (Affinity Biosciences, #DF6765, Rabbit, 1:50), APOD (Proteintech, #10520-1-AP, Rabbit, 1:50), TGFB1 (Affinity Biosciences, #AF1027, Rabbit, 1:100), TGFB3 (Proteintech, #18942-1-AP, Rabbit, 1:50), WNT2B (Affinity Biosciences, #DF12538, Rabbit, 1:100), WNT9A (Affinity Biosciences, #DF9044, Rabbit, 1:100), CD68 (Abcam,ab955,1:3000), CD206 (Cell Signaling,#24595,1:200), and CD163 (Cell Signaling,#93498,1:250) at 4°C overnight.

Techniques: Expressing, Standard Deviation