anti gpihbp1  (Novus Biologicals)

Journal: Bioscience Reports

Article Title: Single-cell RNAseq of Angiotensin II-induced abdominal aortic tissue identifies aneurysm-associated cell clusters in C57BL/6J mice

doi: 10.1042/BSR20241235

Figure Lengend Snippet: ( A ) Stacked column chart shows the proportion of endothelial cell subtypes in the NC, no-AAA, and AAA groups. The proportion of endothelial cell subtypes among the three groups was statistically significant, **** P <0.0001 by Chi-square test. In the NC group, the proportions of malignant, endothelium_EMT, and endothelium were 2.2%, 1.4%, and 96.4%, respectively. In the no-AAA group, the proportions of malignant, endothelium_EMT, and endothelium were 19.0%, 6.9%, and 74.1%, respectively. In the AAA group, the proportions of malignant, endothelium_EMT, and endothelium were 50.4%, 10.2%, and 39.4%, respectively. ( B ) The expression of Cdh5 in 12 clusters of cells from NC, no-AAA, and AAA groups is visualized by feature plots, where three subtypes of epithelial cells can be differentiated. ( C ) The expression of selected marker genes Cd36, Fabp4, Lpl, Gpihbp1 for the malignant cluster from the NC, no-AAA, and AAA groups is visualized through feature plots. ( D ) A dual-plot gene expression heatmap shows the highest co-expression of Lpl and Gpihbp1 in the malignant cluster. ( E ) Representative immunofluorescence images of aorta sections stained with anti-Lpl (red) and anti-Gpihbp1 (green). White arrows indicate the co-expression of Lpl and Gpihbp1. Scale bars, 200 μm. The asterisk symbols indicate the location within the lumen of the abdominal aorta. ( F ) Bar chart of enrichment ratios for Kyoto Encyclopedia of Genes and Genomes (KEGG, upper panel) and Gene Ontology (GO, lower panel) pathways in the malignant cluster of 1601 diferentially expressed genes (including 1168 up-regulated genes and 433 down-regulated genes). False discovery rate (FDR) ≤0.05 indicates significantly enriched pathways, as indicated by the dark blue color. Enrichment ratio = the number of observed genes/ the number of expected genes from each GO or KEGG category in the gene list. ( G ) The expression of Pparg in the 12 clusters from the NC, no-AAA and AAA groups is visualized by feature plots. Note that Pparg is highly expressed in malignant. AAA, abdominal aortic aneurysm; EMT, epithelial-mesenchymal transition; NC, negative control.

Article Snippet: In order to confirm the cell cluster changes, the first antibodies used for immunofluorescence staining, including mouse anti-osteopontin (OPN, also named as SPP1) antibody (1:200, AF808, R&D system, U.S.A.), rabbit anti-CD68 antibody (1:100, 97778, Cell Signaling Technology, U.S.A.), mouse anti-lipoprotein lipase (LPL) antibody (1:200, ab93898, Abcam, U.K.), rabbit anti-GPIHBP1 antibody (1:20, NB110-41539, Novus Biologicals, U.S.A.), rabbit anti-CXCR4 antibody (1:100, T55380S, ABmart, China), and rabbit anti-ACKR3 antibody (1:100, A12712, ABclonal, China).

Techniques: Expressing, Marker, Gene Expression, Immunofluorescence, Staining, Negative Control

Journal: Translational Cancer Research

Article Title: Increase in GPIHBP1 expression in advanced stage colorectal cancer indicates poor immune surveillance

doi: 10.21037/tcr-23-1766

Figure Lengend Snippet: Bioinformatics of GPIHBP1 mRNA in CRC based on TCGA data. (A) The expression of GPIHBP1 mRNA in healthy control and CRC samples. (B) The expression of GPIHBP1 mRNA in tumour tissues and surrounding normal tissues. (C) Survival analysis of GPIHBP1 high and GPIHBP1 low patients, data are presented as HR (95% CI). (D) Variations of GPIHBP1 mRNA in CRC patients of different TNM stages. (E) Enrichment scores of neutrophils and macrophages in GPIHBP1 high and GPIHBP1 low patients. ns, no significance; *, P<0.05; ***, P<0.001. GPIHBP1, GPI-anchored high-density lipoprotein-binding protein 1; GPI, glycosylphosphatidylinositol; HR, hazard ratio; CI, confidence interval; mRNA, messenger RNA; CRC, colorectal cancer; TCGA, The Cancer Genome Atlas; TNM, tumour-node-metastasis.

Article Snippet: Equal amounts of protein (20 µg) were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes; nonspecific sites were blocked with 5% BSA in tris-buffered saline with Tween-20 (TBST) and the membranes were then incubated with anti-GPIHBP1 (1:1,000; Abcam; ab224728), ACC (1:1,000; Cell Signalling Technology; 3676), p-ACC (1:1,000; Cell Signalling Technology; 3661), CPT-1α (1:1,000; Proteintech; 66039-1-Ig), C/EBPβ (1:1,000; Abcam; ab53138), Glut1 (1:1,000; Proteintech; 66290-1-Ig) and anti-β-actin (1:5,000; Servicebio; GB15001); were sequentially incubated with horse radish peroxidase conjugated anti-rabbit or anti-mouse secondary antibody (1:2,000; Invitrogen).

Techniques: Expressing, Control, Binding Assay

Journal: Translational Cancer Research

Article Title: Increase in GPIHBP1 expression in advanced stage colorectal cancer indicates poor immune surveillance

doi: 10.21037/tcr-23-1766

Figure Lengend Snippet: Variations of GPIHBP1 protein levels in CRC. (A) IHC staining results of GPIHBP1 in patients with different stages of CRC (scale bar: 50 µm). (B) Comparison of GPIHBP1 presence in cancer foci or stroma. (C,D) Correlational study of GPIHBP1 presence in cancer foci or stroma with CRC progression. ***, P<0.001. GPIHBP1, GPI-anchored high-density lipoprotein-binding protein 1; GPI, glycosylphosphatidylinositol; CRC, colorectal cancer; IHC, immunohistochemistry.

Article Snippet: Equal amounts of protein (20 µg) were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes; nonspecific sites were blocked with 5% BSA in tris-buffered saline with Tween-20 (TBST) and the membranes were then incubated with anti-GPIHBP1 (1:1,000; Abcam; ab224728), ACC (1:1,000; Cell Signalling Technology; 3676), p-ACC (1:1,000; Cell Signalling Technology; 3661), CPT-1α (1:1,000; Proteintech; 66039-1-Ig), C/EBPβ (1:1,000; Abcam; ab53138), Glut1 (1:1,000; Proteintech; 66290-1-Ig) and anti-β-actin (1:5,000; Servicebio; GB15001); were sequentially incubated with horse radish peroxidase conjugated anti-rabbit or anti-mouse secondary antibody (1:2,000; Invitrogen).

Techniques: Immunohistochemistry, Comparison, Binding Assay

Journal: Translational Cancer Research

Article Title: Increase in GPIHBP1 expression in advanced stage colorectal cancer indicates poor immune surveillance

doi: 10.21037/tcr-23-1766

Figure Lengend Snippet: Macrophages and MDSCs in different stages of CRC. (A) Representative IHC staining results of CD68, Arg1, iNOS, and S100A8 (scale bar: 50 µm). (B) Comparisons of CD68, Arg1, iNOS, or S100A8 presence in patients with different stages of CRC. Correlational study of GPIHBP1 presence in cancer foci (C) or stroma (D) with above myeloid cell markers. ns, no significance; *, P <0.05; **, P<0.01; ***, P<0.001. Arg1, arginase 1; iNOS, inducible nitric oxide synthase; GPIHBP1, GPI-anchored high-density lipoprotein-binding protein 1; GPI, glycosylphosphatidylinositol; MDSC, myeloid-derived suppressor cell; CRC, colorectal cancer; IHC, immunohistochemistry.

Article Snippet: Equal amounts of protein (20 µg) were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes; nonspecific sites were blocked with 5% BSA in tris-buffered saline with Tween-20 (TBST) and the membranes were then incubated with anti-GPIHBP1 (1:1,000; Abcam; ab224728), ACC (1:1,000; Cell Signalling Technology; 3676), p-ACC (1:1,000; Cell Signalling Technology; 3661), CPT-1α (1:1,000; Proteintech; 66039-1-Ig), C/EBPβ (1:1,000; Abcam; ab53138), Glut1 (1:1,000; Proteintech; 66290-1-Ig) and anti-β-actin (1:5,000; Servicebio; GB15001); were sequentially incubated with horse radish peroxidase conjugated anti-rabbit or anti-mouse secondary antibody (1:2,000; Invitrogen).

Techniques: Immunohistochemistry, Binding Assay, Derivative Assay

Journal: Translational Cancer Research

Article Title: Increase in GPIHBP1 expression in advanced stage colorectal cancer indicates poor immune surveillance

doi: 10.21037/tcr-23-1766

Figure Lengend Snippet: CD8 + or CD56 + cells infiltrated in CRC. (A) IHC staining results of CD8, CD56, and GZMB (scale bar: 50 µm). (B) Comparisons of CD8, CD56, or GZMB presence in patients with different stages of CRC. Correlational study of GPIHBP1 presence in cancer foci (C) or stroma (D) with infiltrations of CD8 + , CD56 + , and GZMB + cells. ***, P<0.001. GPIHBP1, GPI-anchored high-density lipoprotein-binding protein 1; GPI, glycosylphosphatidylinositol; CRC, colorectal cancer; IHC, immunohistochemistry; GZMB, granzyme B.

Article Snippet: Equal amounts of protein (20 µg) were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes; nonspecific sites were blocked with 5% BSA in tris-buffered saline with Tween-20 (TBST) and the membranes were then incubated with anti-GPIHBP1 (1:1,000; Abcam; ab224728), ACC (1:1,000; Cell Signalling Technology; 3676), p-ACC (1:1,000; Cell Signalling Technology; 3661), CPT-1α (1:1,000; Proteintech; 66039-1-Ig), C/EBPβ (1:1,000; Abcam; ab53138), Glut1 (1:1,000; Proteintech; 66290-1-Ig) and anti-β-actin (1:5,000; Servicebio; GB15001); were sequentially incubated with horse radish peroxidase conjugated anti-rabbit or anti-mouse secondary antibody (1:2,000; Invitrogen).

Techniques: Immunohistochemistry, Binding Assay

Journal: Translational Cancer Research

Article Title: Increase in GPIHBP1 expression in advanced stage colorectal cancer indicates poor immune surveillance

doi: 10.21037/tcr-23-1766

Figure Lengend Snippet: Ectopic expression of GPIHBP1 promoted tumour growth and immune evasion in vivo . (A) Expression of GPIHBP1 in 293T, MC38, CMT93, and H22 cell lines identified by Western blot. (B) Overexpression of GPIHBP1 in MC38 cells. (C) Variations of key proteins in glycolipid metabolism between MC38-Mock and MC38-GPIHBP1. In vivo tumours formed by MC38-Mock or MC38-GPIHBP1 cells (D,E) (n=6). (F) Growth curve of tumour in tumour-bearing mice. (G) Frequencies of infiltrating immune cells detected by flow cytometry, including CD8 + T cells (CD3 + CD8 + ), NK cells (CD3 − NK1.1 + ), CD4 + T cells (CD3 + CD4 + ), Macrophages (CD11b + F4/80 + ), M1 (CD11b + F4/80 + CD86 + ), M2 (CD11b + F4/80 + CD206 + ) and MDSCs (CD11b + Gr-1 + ). Representative IHC staining results of GPIHBP1, F4/80, S100A8, Arg1, iNOS, and CD8 (scale bar: 20 µm) (H) and respective statistics (I). The experiment was repeated twice. ns, no significance; *, P <0.05; **, P<0.01; ***, P<0.001. GPIHBP1, GPI-anchored high-density lipoprotein-binding protein 1; GPI, glycosylphosphatidylinositol; NK, natural killer; MDSC, myeloid-derived suppressor cell; Arg1, arginase 1; iNOS, inducible nitric oxide synthase; IHC, immunohistochemistry.

Article Snippet: Equal amounts of protein (20 µg) were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes; nonspecific sites were blocked with 5% BSA in tris-buffered saline with Tween-20 (TBST) and the membranes were then incubated with anti-GPIHBP1 (1:1,000; Abcam; ab224728), ACC (1:1,000; Cell Signalling Technology; 3676), p-ACC (1:1,000; Cell Signalling Technology; 3661), CPT-1α (1:1,000; Proteintech; 66039-1-Ig), C/EBPβ (1:1,000; Abcam; ab53138), Glut1 (1:1,000; Proteintech; 66290-1-Ig) and anti-β-actin (1:5,000; Servicebio; GB15001); were sequentially incubated with horse radish peroxidase conjugated anti-rabbit or anti-mouse secondary antibody (1:2,000; Invitrogen).

Techniques: Expressing, In Vivo, Western Blot, Over Expression, Flow Cytometry, Immunohistochemistry, Binding Assay, Derivative Assay

Journal: Journal of Lipid Research

Article Title: A novel NanoBiT-based assay monitors the interaction between lipoprotein lipase and GPIHBP1 in real time

doi: 10.1194/jlr.d119000388

Figure Lengend Snippet: Fig. 1. Association of largeBiT-LPL with smallBiT- GPIHBP1. A: Schematic of the NanoBiT LPL binding assay. B: SmallBiT-GPIHBP1-expressing RHMVECs were grown to confluence. Luminescent substrate (Nano-Glo® live cell substrate) was added to cells and luminescence was read every minute. After 6 min (dotted line), the indicated concentrations of large- BiT-LPL or largeBiT-EL were added to each well and luminescence continued to be measured each minute for an additional 30 min. Points represent mean ± 95% CI of four independent experiments (n = 6 per group per experiment). C: Western blot of the Large- BiT LPL (LPL-LgBiT)- and LargeBiT-EL (EL-LgBiT)- conditioned media used in B. D: Western blot of the LargeBiT LPL (LPL)- and LargeBiT-LPL C445Y (mutLPL)-conditioned media used in E. E: SmallBiT- GPIHBP1-expressing RHMVECs were grown to con- fluence. Luminescent substrate was added to cells and luminescence was read every minute. After 6 min (dotted line), the indicated concentrations of large- BiT-LPL (LPL) or largeBiT-LPL C445Y (mutLPL) were added to each well and luminescence continued to be measured each minute for an additional 30 min. Points represent mean ± 95% CI of three indepen- dent experiments (n = 6 per group per experiment).

Article Snippet: Primary antibody dilutions were 1:5,000 for a mouse monoclonal antibody against FLAG-tag (F1804; Sigma-Aldrich), 1:2,000 for a rat monoclonal antibody against GPIHBP1 [11A12 (42); a kind gift from Loren Fong], and 1:1,000 for a goat antibody against actin (SC-1615; Santa Cruz).

Techniques: Binding Assay, Expressing, Western Blot

Journal: Journal of Lipid Research

Article Title: A novel NanoBiT-based assay monitors the interaction between lipoprotein lipase and GPIHBP1 in real time

doi: 10.1194/jlr.d119000388

Figure Lengend Snippet: Fig. 2. Disruption of LPL-GPIHBP1 binding by hep- arin. A: Schematic of the NanoBiT LPL dissociation assay. B: Western blot of lysates of smallBiT-GPIHBP1- expressing RHMVECs incubated with LargeBiT-LPL for 3.5 h at 4°C, washed, and then treated with or with- out 100 U/ml heparin for 30 min. C, D: SmallBiT- GPIHBP1-expressing endothelial cells were incubated with LargeBiT-LPL for 2 h at 4°C and washed. Lumi- nescent substrate was added to cells and luminescence read every 3 min for 15 min. After 15 min 100 U/ml heparin (+heparin) or water (–heparin) were added to the samples and luminescence was read every 3 min for an additional 15 min. C: Luminescent signal over time (maximum signal for each sample set to 100%). D: Luminescence signal over time normalized to the –heparin control at each time point. Points repre- sent mean ± 95% CI of three independent experiments (n = 3–6 per group per experiment).

Article Snippet: Primary antibody dilutions were 1:5,000 for a mouse monoclonal antibody against FLAG-tag (F1804; Sigma-Aldrich), 1:2,000 for a rat monoclonal antibody against GPIHBP1 [11A12 (42); a kind gift from Loren Fong], and 1:1,000 for a goat antibody against actin (SC-1615; Santa Cruz).

Techniques: Disruption, Binding Assay, Western Blot, Expressing, Incubation, Control

Journal: Journal of Lipid Research

Article Title: A novel NanoBiT-based assay monitors the interaction between lipoprotein lipase and GPIHBP1 in real time

doi: 10.1194/jlr.d119000388

Figure Lengend Snippet: Fig. 3. Disruption of LPL-GPIHBP1 binding by ANGPTL proteins. A: RHMVECs expressing smallBiT- GPIHBP1 were incubated with largeBiT-LPL for 2 h at 4°C and washed. Luminescent substrate was added to cells and luminescence read every 3 min for 15 min. After 15 min (dotted line), the indicated ANGPTL proteins were added and luminescence continued to be measured every 3 min for 45 min. Points represent mean ± 95% CI of luminescent signal over time nor- malized to the “LPL only” control at each time point. Data represent seven independent experiments with three biological replicates per group. B: Western blot of cell lysates from cells incubated with largeBiT-LPL for 3.5 h at 4°C, washed, and then treated with ANG- PTL4, ANGPTL3, ANGPTL8, or heparin for 30 min at 37°C. Bands show LPL (using an antibody against the FLAG tag), GPIHBP1, and actin for biological tripli- cates. C: LPL activity of LPL bound to smallBiT-GPI- HBP1-expressing RHMVECs washed and treated with ANGPTL4 (A4), ANGPTL3 (A3), ANGPTL8 (A8), or ANGPTL3 and ANGPTL8 (A3/8) for 30 min at 37°C. After incubation, cells were washed, and LPL was re- leased from the cells with heparin before performing LPL activity assays. Bars represent normalized LPL ac- tivity (mean ± 95% CI of three independent experi- ments; n = 3 per experiment). D: Luminescence of smallBiT-GPIHBP1-expressing RHMVECs incubated with largeBiT-LPL. After washing off unbound LPL, luminescence was measured for 15 min at 3 min inter- vals. The indicated ANGPTL proteins were added (first dotted line) and luminescence was measured for an additional 30 min. Cells were then washed, fresh substrate was added (second dotted line), and lumi- nescence was measured for another 15 min. Points represent luminescent signal normalized to the LPL only control at each time point (mean ± 95% CI of four independent experiments; n = 3 per group per experiment).

Article Snippet: Primary antibody dilutions were 1:5,000 for a mouse monoclonal antibody against FLAG-tag (F1804; Sigma-Aldrich), 1:2,000 for a rat monoclonal antibody against GPIHBP1 [11A12 (42); a kind gift from Loren Fong], and 1:1,000 for a goat antibody against actin (SC-1615; Santa Cruz).

Techniques: Disruption, Binding Assay, Expressing, Incubation, Control, Western Blot, FLAG-tag, Activity Assay

Journal: Journal of Lipid Research

Article Title: A novel NanoBiT-based assay monitors the interaction between lipoprotein lipase and GPIHBP1 in real time

doi: 10.1194/jlr.d119000388

Figure Lengend Snippet: Fig. 4. Effect of THL on GPIHBP1-LPL binding. A: RHMVECs expressing smallBiT-GPIHBP1 were incubated with largeBiT-LPL for 2 h at 4°C and washed. Luminescent substrate was added to the cells, and luminescence was read every 3 min for 15 min. After 15 min (dotted line), 0–80 M of THL were added and luminescence continued to be measured every 3 min for 45 min. Points represent mean ± 95% CI of luminescent signal over time normalized to the vehicle (ethanol). Data represent three independent experiments each with three biological replicates per group. B: Western blot of cell lysates from cells incubated with largeBiT-LPL for 3.5 h at 4°C, washed, and then treated with 80 M of THL or 100 U/ml heparin for 30 min. Bands show LPL (using an antibody against the FLAG tag), GPIHBP1, and actin for biological triplicates. C, D: LPL activity of largeBiT-LPL bound to smallBiT-GPIHBP1-expressing RHMVECs (C) or FLAG-LPL bound to S tag-GPIHBP1 (D) after treatment with 80 M of THL for 30 min at 37°C. After incubation,

Article Snippet: Primary antibody dilutions were 1:5,000 for a mouse monoclonal antibody against FLAG-tag (F1804; Sigma-Aldrich), 1:2,000 for a rat monoclonal antibody against GPIHBP1 [11A12 (42); a kind gift from Loren Fong], and 1:1,000 for a goat antibody against actin (SC-1615; Santa Cruz).

Techniques: Binding Assay, Expressing, Incubation, Western Blot, FLAG-tag, Activity Assay

Journal: Journal of Lipid Research

Article Title: A novel NanoBiT-based assay monitors the interaction between lipoprotein lipase and GPIHBP1 in real time

doi: 10.1194/jlr.d119000388

Figure Lengend Snippet: Fig. 6. Effect of THL on chylomicron-LPL interactions. A: RHM- VECs expressing smBiT-GPIHBP1 were incubated with LargeBiT- LPL for 2 h at 4°C and washed. Cells were then treated with 80 M of THL or vehicle for 30 min at 37°C. After washing, luminescent substrate was added to cells and luminescence read every 3 min for 12 min. After 12 min, chylomicrons (Chylo) (0–20 g/ml by pro- tein) were added and luminescence continued to be measured for 33 min. Points represent mean ± 95% CI of luminescent signal over time normalized to control at each time point. Data represent three independent experiments, each with 3–6 biological replicates per group. B: Immunofluorescence showing binding of LPL and chylo- microns to smallBiT-GPIHBP1-expressing RHMVECs. RHMVECs were incubated with largeBiT-LPL for 3 h at 4°C. After washing away unbound LPL, cells were incubated with or without THL (80 M) for 30 min at 37°C. Cells were then washed and incubated with or without fluorescently labeled chylomicrons (green) for 30 min at 37°C. Cells were then stained for LPL (red) using an antibody against the FLAG tag and with DAPI (blue).

Article Snippet: Primary antibody dilutions were 1:5,000 for a mouse monoclonal antibody against FLAG-tag (F1804; Sigma-Aldrich), 1:2,000 for a rat monoclonal antibody against GPIHBP1 [11A12 (42); a kind gift from Loren Fong], and 1:1,000 for a goat antibody against actin (SC-1615; Santa Cruz).

Techniques: Expressing, Incubation, Control, Immunofluorescence, Binding Assay, Labeling, Staining, FLAG-tag

Journal: Journal of Lipid Research

Article Title: A novel NanoBiT-based assay monitors the interaction between lipoprotein lipase and GPIHBP1 in real time

doi: 10.1194/jlr.d119000388

Figure Lengend Snippet: Fig. 5. Effect of chylomicrons and fatty acids on GPIHBP1-LPL binding. RHMVECs expressing smBiT-GPIHBP1 were incubated with LargeBiT-LPL for 2 h at 4°C and washed. Luminescent sub- strate was added to cells and luminescence read every 3 min for 12–15 min. A: After 15 min (dotted line), chylomicrons (chylo) (1–10 g/ml by protein) were added and luminescence continued to be measured for 45 min. ANGPTL4 (90 ng/ml) was also added as a control. B: After 12 min, chylomicrons or heparin were added (first dotted line) at the indicated concentrations and lumines- cence was measured for an additional 12 min. Cells were then washed, fresh substrate was added (second dotted line), and lumi- nescence was measured for another 15 min. C: After 15 min (dotted line), sodium oleate (0.375–3 mM) was added and luminescence continued to be measured for 45 min. For all panels, points repre- sent luminescent signal over time normalized to control at each time point (mean ± 95% CI of three independent experiments; n = 3–6 per group per experiment).

Article Snippet: Primary antibody dilutions were 1:5,000 for a mouse monoclonal antibody against FLAG-tag (F1804; Sigma-Aldrich), 1:2,000 for a rat monoclonal antibody against GPIHBP1 [11A12 (42); a kind gift from Loren Fong], and 1:1,000 for a goat antibody against actin (SC-1615; Santa Cruz).

Techniques: Binding Assay, Expressing, Incubation, Control

Journal: Journal of Lipid Research

Article Title: A novel NanoBiT-based assay monitors the interaction between lipoprotein lipase and GPIHBP1 in real time

doi: 10.1194/jlr.d119000388

Figure Lengend Snippet: Fig. 7. Effect of tyloxapol and P-407 on GPIHBP1-LPL binding and LPL activity. A, B: RHMVECs expressing smallBiT-GPIHBP1 were incu- bated with largeBiT-LPL for 2 h at 4°C and washed. Luminescent substrate was added to cells and luminescence read every 3 min for 15 min. After 15 min (dotted line), 150–600 g/ml of tyloxapol (tylox) (A) or 2–8 mg/ml of P-407 (B) were added and luminescence continued to be measured for 45 min. Points represent mean ± 95% CI of luminescent signal over time normalized to the control at each time point. Data represent three independent experiments, each with three biological triplicates per group. C, D: Western blot of cell lysates from cells incu- bated with LargeBiT-LPL for 3.5 h at 4°C, washed, and then treated with 8 mg/ml tyloxapol (C) or 8 mg/ml P-407 (D) for 30 min at 37°C.

Article Snippet: Primary antibody dilutions were 1:5,000 for a mouse monoclonal antibody against FLAG-tag (F1804; Sigma-Aldrich), 1:2,000 for a rat monoclonal antibody against GPIHBP1 [11A12 (42); a kind gift from Loren Fong], and 1:1,000 for a goat antibody against actin (SC-1615; Santa Cruz).

Techniques: Binding Assay, Activity Assay, Expressing, Control, Western Blot

Journal: Journal of Lipid Research

Article Title: A novel NanoBiT-based assay monitors the interaction between lipoprotein lipase and GPIHBP1 in real time

doi: 10.1194/jlr.d119000388

Figure Lengend Snippet: Fig. 8. Effect of tyloxapol on binding of chylomicrons to LPL. A: Chylomicrons (20 g/ml) were mixed with 2 or 8 mg/ml tyloxapol (tylox) and then incubated with largeBiT-LPL bound to smallBiT- GPIHBP1-expressing RHMVECs at 37°C. After 30 min, cells were washed, substrate was added, and luminescence was read. Bars repre- sent luminescent signal (mean ± 95% CI of four independent ex- periments; n = 4 per group per experiment) normalized to the no chylomicron no tyloxapol control. B: Immunofluorescence showing binding of LPL and chylomicrons (chylos) to smallBiT-GPIHBP1- expressing RHMVECs. RHMVECs were incubated with largeBiT-LPL

Article Snippet: Primary antibody dilutions were 1:5,000 for a mouse monoclonal antibody against FLAG-tag (F1804; Sigma-Aldrich), 1:2,000 for a rat monoclonal antibody against GPIHBP1 [11A12 (42); a kind gift from Loren Fong], and 1:1,000 for a goat antibody against actin (SC-1615; Santa Cruz).

Techniques: Binding Assay, Incubation, Expressing, Control, Immunofluorescence

Journal: eLife

Article Title: Defining the role of pulmonary endothelial cell heterogeneity in the response to acute lung injury

doi: 10.7554/eLife.53072

Figure Lengend Snippet: ( A ) Circos plot shows gene expression and gene ontology (GO) overlap between distinct clusters of pulmonary ECs. A purple line connecting two clusters indicates expression of the same gene in both clusters, while a blue line connecting two clusters indicates expression of different genes found within the same GO category in each cluster. ( B ) GO biological process enrichment performed for each cluster and displayed in heatmap format demonstrates expression of genes associated with different biological processes in each cluster, including some overlap between clusters. Cluster 2 (green box) shares enrichment of some processes related to angiogenesis and blood vessel development with cluster 0 (blue box) but is distinct in its enrichment of genes related to vasculogenesis. ( C ) In-depth analysis of gene expression in cluster four indicates that this cluster likely represents macrovascular ECs (maECs) with high expression of Vwf and Vcam1 . IHC indicates that these proteins localize mainly to the large vessel endothelium. White arrowhead demonstrates Vcam1 located in nearby mesenchymal cells. ( D ) Clusters 0, 1, and three represent a heterogeneous population of microvascular ECs (miECs) with high expression of Gpihbp1 and Plvap . IHC indicates that these proteins localize to the alveolar capillary plexus endothelium. Yellow arrowhead demonstrates Plvap present in the large vessel endothelium. ( E ) Cluster two represents an as-yet uncharacterized population of ECs that localize to the alveolar region and express surface marker Cd34 at a higher level from that in other ECs. These cells also express high levels of Car4 . CD34 and Car4 proteins localize to the alveolar space, indicating a similar spatial distribution of cells in cluster two to that of miECs. v, vessel; a, alveolar space; scale bars in ( C )-( E ), 20 microns.

Article Snippet: Antibody , anti-Gpihbp1 (rabbit polyclonal) , Thermo Fisher , PA1-16976; RRID: AB_2294825 , IHC (1:100).

Techniques: Expressing, Marker

Journal: eLife

Article Title: Defining the role of pulmonary endothelial cell heterogeneity in the response to acute lung injury

doi: 10.7554/eLife.53072

Figure Lengend Snippet: ( A ) RNAscope for Gpihbp1 indicates that miECs, which also express Pecam1 and Plvap (blue outlines), are located throughout the alveolar space. Gpihbp1 -expressing cells are not found within large vessels, indicated by orange dotted lines. Cells within large vessels express Plvap and Pecam1 (yellow outlines). ( B, B’ ) RNAscope for Cxcl12 , a marker for arterial maECs, indicates that these cells ( Cxcl12 + / Pecam1 + , blue outlines) localize to large vessels indicated by orange dotted lines but not to the alveolar capillary space at homeostasis. Pecam1 + / Cxcl12 - cells (yellow outlines) are found outside of the large vessels. ( C, C’ ) RNAscope for Vegfc , a marker of venous maECs, localizes these cells to large vessels at homeostasis ( Vegfc + / Pecam1 + , blue outlines). Pecam1 + / Vegfc - cells (yellow outlines) are found outside of the large vessels. ( D ) At 14 days post-H1N1 influenza injury, Gpihbp1 / Pecam1 / Plvap triple-positive miECs are still localized to the alveolar capillary space. ( E, E’ ) After H1N1 injury, Cxcl12 + / Pecam1 + arterial maECs continue to be present in large vessels, but not in the capillary endothelium. ( F, F’ ) Vegfc + / Pecam1 + venous maECs continue to localize to large vessels after H1N1 influenza injury. Scale bars in ( A ), ( B ), ( C ), ( D ), ( E ), and ( F ), 100 microns. Scale bars in ( A’–A’’’ ), ( B’ ), ( C’ ), ( D’–D’’’ ), ( E’ ), and ( F’ ), 50 microns.

Article Snippet: Antibody , anti-Gpihbp1 (rabbit polyclonal) , Thermo Fisher , PA1-16976; RRID: AB_2294825 , IHC (1:100).

Techniques: Expressing, Marker

Journal: eLife

Article Title: Defining the role of pulmonary endothelial cell heterogeneity in the response to acute lung injury

doi: 10.7554/eLife.53072

Figure Lengend Snippet: ( A ) UMAP dimension reduction of H1N1 injury scRNA-seq dataset, with red box enclosing EC clusters and representing areas of increased digital zoom displayed in ( B )-( I ). Black circle demarcates proliferating EC cluster. Proliferating ECs express genes highly expressed in all ECs or in miECs, such as ( B ) Cd31 , ( C ) Gpihbp1 , and ( D ) Plvap . However, they express moderate levels of ( E ) Cd34 and do not express other genes that are highly expressed in Car4 -high ECs, such as ( F ) Kdr or ( G ) Ednrb . Proliferating ECs do not express genes enriched in maECs, such as ( H ) Vcam1 or ( I ) Vwf.

Article Snippet: Antibody , anti-Gpihbp1 (rabbit polyclonal) , Thermo Fisher , PA1-16976; RRID: AB_2294825 , IHC (1:100).

Techniques:

Journal: eLife

Article Title: Defining the role of pulmonary endothelial cell heterogeneity in the response to acute lung injury

doi: 10.7554/eLife.53072

Figure Lengend Snippet: ( A ) Pseudotime analysis of Car4 -high EC, Gpihbp1 -high miEC, Ifi47 -high miEC, and proliferating EC clusters at 14 dpi using Slingshot identifies putative lineage relationships between the clusters based on gene expression similarities. Proliferating ECs are more similar in gene expression to both miEC populations than to Car4 -high ECs. ( B ) Proliferating ECs have the highest G2/M score among all four populations, with orange/red indicating cells predicted to be in G2/M and blue indicating cells not predicted to be in G2/M. ( C ), ( D ) Slingshot identifies two lineage trajectories among the four EC populations. Curve one connects Car4 -high ECs and proliferating ECs through a miEC intermediate, while curve two connects Car4 -high ECs and miECs. Color indicates direction of the trajectory, which is defined by the program and proceeds from blue to red. ( E ), ( F ) Heatmaps displaying the top 50 differentially expressed genes along curves 1 and 2. Curve one demonstrates the greatest variance in gene expression along the trajectory, indicating greater gene expression differences between Car4 -high ECs and proliferating ECs than between Car4 -high ECs and miECs.

Article Snippet: Antibody , anti-Gpihbp1 (rabbit polyclonal) , Thermo Fisher , PA1-16976; RRID: AB_2294825 , IHC (1:100).

Techniques: Expressing

Journal: eLife

Article Title: Defining the role of pulmonary endothelial cell heterogeneity in the response to acute lung injury

doi: 10.7554/eLife.53072

Figure Lengend Snippet: Primer sequences.

Article Snippet: Antibody , anti-Gpihbp1 (rabbit polyclonal) , Thermo Fisher , PA1-16976; RRID: AB_2294825 , IHC (1:100).

Techniques: Sequencing

Journal: eLife

Article Title: Defining the role of pulmonary endothelial cell heterogeneity in the response to acute lung injury

doi: 10.7554/eLife.53072

Figure Lengend Snippet:

Article Snippet: Antibody , anti-Gpihbp1 (rabbit polyclonal) , Thermo Fisher , PA1-16976; RRID: AB_2294825 , IHC (1:100).

Techniques: Flow Cytometry