INSL5 Antibody - BSA Free

Western Blot: INSL5 Antibody - BSA Free [NBP1-86343]

Analysis in control (vector only transfected HEK293T lysate) and INSL5 over-expression lysate (Co-expressed with a C-terminal myc-DDK tag (~3.1 kDa) in mammalian HEK293T cells).

Western Blot: INSL5 Antibody - BSA Free [NBP1-86343] -

INSL5 induces glucose metabolism to aerobic glycolysis reprogramming in NPC cellsAAnalysis of glycolytic gene expression by qRT–PCR in INSL5 overexpressed CNE1 cell lines.BAnalysis of glycolytic gene expression by immunoblot in INSL5 overexpressing cells with or without GPCR142 knockdown.C–EThe metabolomics identified increased glycolytic intermediate metabolites in INSL5 overexpressed CNE1 cells (C), CNE2 cells (D), and HK1 cells (E). CNE1 and HK1 were from five independent samples, and CNE2 were from seven independent samples.FSchematic diagram of aerobic glycolysis pathway and TCA pathway. Red: INSL5 upregulated glycolytic genes and metabolites. Green: INSL5 downregulated TCA intermediates.GThe extracellular acidification rate (ECAR) was measured in cells with or without INSL5 overexpression using a Seahorse XF96 Extracellular Flux analyzer.HThe oxygen consumption rate (OCR) was measured in cells with or without INSL5 overexpression using a Seahorse XF96 Extracellular Flux analyzer.I–LGlucose uptake (I), HK2 enzyme activity (J), ATP concentration (K), and lactate production (L) in CNE2 and HK1 stable cells.MGlucose uptake in CNE2 wide‐type or GPCR142 knockdown cells stimulated with INSL5 peptide (50 ng/ml) for 24 h.NATP concentration, HK2 enzyme activity, and lactate production in INSL5 wide‐type or knockdown CNE2‐EBV cells.Data information: In (G–I, M and N), data are presented as mean +/- SEM, in (C–E and J–L), data are presented as mean +/- SD, from three different experiments, and P‐values were determined by unpaired t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance. Exact P‐values are specified in Appendix Table S4.Source data are available online for this figure. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/32657028), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Flow Cytometry: INSL5 Antibody - BSA Free [NBP1-86343] -

Overexpression of INSL5 promotes cell cycle progression and suppresses cell apoptosisAThe cell cycle of vector control or INSL5 overexpressing CNE1, CNE2, and HK1 cells were analyzed by flow cytometry assay.BStatistical analysis of cell percentage in each cell cycle phase.CCyclin D, cyclin E, cyclin B, and p27 expression levels were detected by Western blotting in cells with or without INSL5 overexpression.DWestern blotting for c‐myc, BCL2, and BCL‐xL in CNE1, CNE2, and HK1 with or without INSL5 overexpression.ECNE1 stable cell line was treated with DDP or 5‐FU and stained with annexin V/propidium iodide (PI), and measured by flow cytometry. Data shown are representative of three independent experiments.FStatistical analysis of the effects of INSL5 overexpression on cell apoptosis under DDP or 5‐FU treatment.GWestern blotting for apoptosis pathway in CNE1, CNE2, and HK1 with or without INSL5 overexpression under 5‐FU treatment.Data information: In (F), data are presented as mean +/- SEM, from three different experiments, and P‐values were determined by unpaired t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance. Exact P‐values are specified in Appendix Table S4.Source data are available online for this figure. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/32657028), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: INSL5 Antibody - BSA Free [NBP1-86343] -

INSL5 promotes the progression of NPC via accelerating cell proliferation and invasion depending on GPCR142AExogenous expression of INSL5 in NPC cells. Representative immunoblotting (upper panel) and immunofluorescent staining (lower panel) showed stable exogenous expression of INSL5 in both CNE1, CNE2, and HK1 NPC cell lines. Scale bars represent 20 μm.BMTT assay of vector control or INSL5 overexpressing CNE1, CNE2, and HK1 NPC cell lines (upper panel) either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2) (lower panel). n = 4 biological replicates for CNE1 and CNE2 cell line, n = 6 biological replicates for HK1.C–HColony formation (C and D), Brdu incorporation (E and F), and migration assays (G and H) of vector control or INSL5 overexpressing CNE1, CNE2, and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (C), (E), and (G) for colony formation, Brdu incorporation, and migration assays, respectively. Number of colonies, the percentage of Brdu positive cells, and migrated cells per field of view were plotted in (D, F, and H), respectively. The results are from three different experiments. Scale bars represent 20 μm in (E) and 100 μm in (G)I, JXenograft tumor growth of INSL5 overexpression NPC HK1 stable cell lines in nude mice. Tumor size (I) and tumor weight (J) of two groups. n = 11 mice per group.Data information: In (B, I, and J), data are presented as mean +/- SD, in (D, F, and H), data are presented as mean +/- SEM, from three different experiments, and P‐values were determined by unpaired t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance. Exact P‐values are specified in Appendix Table S4.Source data are available online for this figure. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/32657028), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: INSL5 Antibody - BSA Free [NBP1-86343] -

INSL5 promotes the progression of NPC via accelerating cell proliferation and invasion depending on GPCR142AExogenous expression of INSL5 in NPC cells. Representative immunoblotting (upper panel) and immunofluorescent staining (lower panel) showed stable exogenous expression of INSL5 in both CNE1, CNE2, and HK1 NPC cell lines. Scale bars represent 20 μm.BMTT assay of vector control or INSL5 overexpressing CNE1, CNE2, and HK1 NPC cell lines (upper panel) either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2) (lower panel). n = 4 biological replicates for CNE1 and CNE2 cell line, n = 6 biological replicates for HK1.C–HColony formation (C and D), Brdu incorporation (E and F), and migration assays (G and H) of vector control or INSL5 overexpressing CNE1, CNE2, and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (C), (E), and (G) for colony formation, Brdu incorporation, and migration assays, respectively. Number of colonies, the percentage of Brdu positive cells, and migrated cells per field of view were plotted in (D, F, and H), respectively. The results are from three different experiments. Scale bars represent 20 μm in (E) and 100 μm in (G)I, JXenograft tumor growth of INSL5 overexpression NPC HK1 stable cell lines in nude mice. Tumor size (I) and tumor weight (J) of two groups. n = 11 mice per group.Data information: In (B, I, and J), data are presented as mean +/- SD, in (D, F, and H), data are presented as mean +/- SEM, from three different experiments, and P‐values were determined by unpaired t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance. Exact P‐values are specified in Appendix Table S4.Source data are available online for this figure. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/32657028), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: INSL5 Antibody - BSA Free [NBP1-86343] -

INSL5 promotes the progression of NPC via accelerating cell proliferation and invasion depending on GPCR142AExogenous expression of INSL5 in NPC cells. Representative immunoblotting (upper panel) and immunofluorescent staining (lower panel) showed stable exogenous expression of INSL5 in both CNE1, CNE2, and HK1 NPC cell lines. Scale bars represent 20 μm.BMTT assay of vector control or INSL5 overexpressing CNE1, CNE2, and HK1 NPC cell lines (upper panel) either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2) (lower panel). n = 4 biological replicates for CNE1 and CNE2 cell line, n = 6 biological replicates for HK1.C–HColony formation (C and D), Brdu incorporation (E and F), and migration assays (G and H) of vector control or INSL5 overexpressing CNE1, CNE2, and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (C), (E), and (G) for colony formation, Brdu incorporation, and migration assays, respectively. Number of colonies, the percentage of Brdu positive cells, and migrated cells per field of view were plotted in (D, F, and H), respectively. The results are from three different experiments. Scale bars represent 20 μm in (E) and 100 μm in (G)I, JXenograft tumor growth of INSL5 overexpression NPC HK1 stable cell lines in nude mice. Tumor size (I) and tumor weight (J) of two groups. n = 11 mice per group.Data information: In (B, I, and J), data are presented as mean +/- SD, in (D, F, and H), data are presented as mean +/- SEM, from three different experiments, and P‐values were determined by unpaired t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance. Exact P‐values are specified in Appendix Table S4.Source data are available online for this figure. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/32657028), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: INSL5 Antibody - BSA Free [NBP1-86343] -

INSL5 promotes the progression of NPC via accelerating cell proliferation and invasion depending on GPCR142AExogenous expression of INSL5 in NPC cells. Representative immunoblotting (upper panel) and immunofluorescent staining (lower panel) showed stable exogenous expression of INSL5 in both CNE1, CNE2, and HK1 NPC cell lines. Scale bars represent 20 μm.BMTT assay of vector control or INSL5 overexpressing CNE1, CNE2, and HK1 NPC cell lines (upper panel) either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2) (lower panel). n = 4 biological replicates for CNE1 and CNE2 cell line, n = 6 biological replicates for HK1.C–HColony formation (C and D), Brdu incorporation (E and F), and migration assays (G and H) of vector control or INSL5 overexpressing CNE1, CNE2, and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (C), (E), and (G) for colony formation, Brdu incorporation, and migration assays, respectively. Number of colonies, the percentage of Brdu positive cells, and migrated cells per field of view were plotted in (D, F, and H), respectively. The results are from three different experiments. Scale bars represent 20 μm in (E) and 100 μm in (G)I, JXenograft tumor growth of INSL5 overexpression NPC HK1 stable cell lines in nude mice. Tumor size (I) and tumor weight (J) of two groups. n = 11 mice per group.Data information: In (B, I, and J), data are presented as mean +/- SD, in (D, F, and H), data are presented as mean +/- SEM, from three different experiments, and P‐values were determined by unpaired t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance. Exact P‐values are specified in Appendix Table S4.Source data are available online for this figure. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/32657028), licensed under a CC-BY license. Not internally tested by Novus Biologicals.