relevant primary antibodies against lbh  (Proteintech)

Journal: EBioMedicine

Article Title: Overexpression of Limb-Bud and Heart (LBH) promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia.

doi: 10.1016/j.ebiom.2019.09.037

Figure Lengend Snippet: Fig. 3. HIF1 can directly induce the expression of LBH under hypoxia a: LBH mRNA expression of T98G (left) and GSC4B (right) gradually increased during prolonged treatment under hypoxia as measured by qPCR. (T98G: p <0.0001, GSC4B: p <0.0001, One-Way ANOVA) b: LBH protein expression of T98G and GSC4B was gradually increased during prolonged treatment under hypoxia as measured by western blotting. c: Sequence motif representing the consensus HIF-1 binding motif (JASPAR database). d, e: Luciferase reporter assays showed hypoxia can upregulate the luciferase promoter activities of LBH in T98G (left) and GSC4B (right) cells. (T98G: p <0.0001, GSC4B: p <0.0001, Student’s t-test) e: ChIP qPCR showed HIF-1 binding to the promoter of LBH under hypoxia. (T98G: p <0.0001, GSC4B: p <0.0001, Student’s t-test) g, h: qPCR (g) and western blot (h) showed the expression of HIF-1 overexpression can upregulate the expression of LBH. (T98G: p <0.0001, GSC4B: p <0.0001, Student’s t-test) All data are shown as the mean ± SD (three independent experiments). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Article Snippet: Relevant primary antibodies against LBH (1:500, #ab122223), HIF-1 (1:500, #ab1), VEGFA (1:1000, #ab52917), VEGFR2 (1:1000, #ab11939), p-VEGFR2 (1:1000, #ab194806), MEK1/2 (1:1000, #ab178876), p-MEK1/2 (1:1000, #ab96379), ERK1/2 (1:500, #ab17942), p-ERK1/2 (1:500, #ab223500) and βactin (1:2000, #66009–1-Ig, ProteinTech, Chicago, IL, USA) were incubated with the membrane at 4 °C overnight.

Techniques: Expressing, Western Blot, Sequencing, Binding Assay, Luciferase, ChIP-qPCR, Over Expression

Journal: EBioMedicine

Article Title: Overexpression of Limb-Bud and Heart (LBH) promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia.

doi: 10.1016/j.ebiom.2019.09.037

Figure Lengend Snippet: Fig. 5. LBH can activate VEGFA-mediated ERK signalling in hBMECs under hypoxia a, b: The VEGFA mRNA expression and secretion level of U118 and GSC2A cells was upregulated under hypoxia and further upregulated after LBH overexpression, as measured by qPCR (a) and ELISA (b). (qPCR: U118: p <0.0001, GSC2A: p <0.0001; ELISA: U118: p = 0.0019, GSC2A: p = 0.0011, One-Way ANOVA) c, d: The VEGFA mRNA expression and secretion level of T98G and GSC4B cells was upregulated under hypoxia and decreased after LBH knockdown, as measured by qPCR (c) and ELISA (d). (qPCR: T98G: p <0.0001, GSC4B: p = 0.0012; ELISA: T98G: p = 0.0023, GSC4B: p = 0.0029, One-Way ANOVA) e: The VEGFA protein expression of U118 and GSC2A cells was upregulated under hypoxia and further upregulated after LBH overexpression, as shown by western blotting. f: The VEGFA protein expression of T98G and GSC4B cells was upregulated under hypoxia and decreased after LBH knockdown, as shown by western blotting. g: The VEGFR-ERK signalling pathway in vascular endothelial cells following treatment with LBH-silenced T98G and GSC4B conditioned media under normoxia or hypoxia was measured by western blotting. All data are shown as the mean ± SD (three independent experiments). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Article Snippet: Relevant primary antibodies against LBH (1:500, #ab122223), HIF-1 (1:500, #ab1), VEGFA (1:1000, #ab52917), VEGFR2 (1:1000, #ab11939), p-VEGFR2 (1:1000, #ab194806), MEK1/2 (1:1000, #ab178876), p-MEK1/2 (1:1000, #ab96379), ERK1/2 (1:500, #ab17942), p-ERK1/2 (1:500, #ab223500) and βactin (1:2000, #66009–1-Ig, ProteinTech, Chicago, IL, USA) were incubated with the membrane at 4 °C overnight.

Techniques: Expressing, Over Expression, Enzyme-linked Immunosorbent Assay, Knockdown, Western Blot

Journal: EBioMedicine

Article Title: Overexpression of Limb-Bud and Heart (LBH) promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia.

doi: 10.1016/j.ebiom.2019.09.037

Figure Lengend Snippet: Fig. 6. Anti-VEGFA treatment can abolish LBH-induced hBMECs proliferation, invasion and angiogenesis under hypoxia a, b: The induction of vascular endothelial cell viability following treatment with LBH-overexpressed U118 (a) and GSC2A (b) conditioned media was reversed following anti-VEGFA treatment, as measured by an MTS assay. (U118: p = 0.0016, GSC2A: p = 0.0011, One-Way ANOVA) c: The proliferation of vascular endothelial cells following treatment with LBH-overexpressed U118 and GSC2A conditioned media was reversed following anti-VEGFA treat- ment, as measured by an EDU incorporation assay. Scale bar = 100 μm. (U118: p = 0.0008, GSC2A: p < 0.0001, One-Way ANOVA) d: Representative transwell assay showing that treatment with LBH-overexpressed U118 and GSC2A conditioned media induced invasion of vascular endothelial cells that was reversed after anti-VEGFA treatment. Scale bar = 100 μm. (U118: p <0.0001, GSC2A: p = 0.0018, One-Way ANOVA) e: Representative tube formation assay showing that treatment with LBH-overexpressed U118 and GSC2A conditioned media induced tubulogenesis of vascular endothelial cells that was reversed after anti-VEGFA treatment. Scale bar = 100 μm. (number of branches: U118: p <0.0001, GSC2A: p < 0.0001, tubule length: U118: p = 0.0012, GSC2A: p = 0.0019, One-Way ANOVA) All data are shown as the mean ± SD (three independent experiments). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Article Snippet: Relevant primary antibodies against LBH (1:500, #ab122223), HIF-1 (1:500, #ab1), VEGFA (1:1000, #ab52917), VEGFR2 (1:1000, #ab11939), p-VEGFR2 (1:1000, #ab194806), MEK1/2 (1:1000, #ab178876), p-MEK1/2 (1:1000, #ab96379), ERK1/2 (1:500, #ab17942), p-ERK1/2 (1:500, #ab223500) and βactin (1:2000, #66009–1-Ig, ProteinTech, Chicago, IL, USA) were incubated with the membrane at 4 °C overnight.

Techniques: MTS Assay, Transwell Assay, Tube Formation Assay

Journal: EBioMedicine

Article Title: Overexpression of Limb-Bud and Heart (LBH) promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia.

doi: 10.1016/j.ebiom.2019.09.037

Figure Lengend Snippet: Fig. 7. LBH regulates glioma tumorigenesis and angiogenesis in vivo a, c: Representative photographs showing the size of intracranial tumors in the coronal position. LBH overexpression in GSC2A cells increased the intracranial tumor size (a), whereas LBH knockdown in GSC4B cells decreased the intracranial tumor size (c). Scale bar = 10 mm. b, d: LBH-overexpressed GSC2A cells implanted into tumor-bearing mice showed shorter survival times as measured by Kaplan–Meier survival curves (b), compared with longer survival times when LBH-silenced GSC4B cells were implanted into tumor bearing mice (d). For each group, n = 5. e: Representative immunohistochemical staining showing the changes in LBH, VEGFA and CD31 in LBH overexpression and knockdown orthotopic xenograft models. Scale bar = 50 μm. f: Schematic diagram to illustrate that overexpression of LBH promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia.

Article Snippet: Relevant primary antibodies against LBH (1:500, #ab122223), HIF-1 (1:500, #ab1), VEGFA (1:1000, #ab52917), VEGFR2 (1:1000, #ab11939), p-VEGFR2 (1:1000, #ab194806), MEK1/2 (1:1000, #ab178876), p-MEK1/2 (1:1000, #ab96379), ERK1/2 (1:500, #ab17942), p-ERK1/2 (1:500, #ab223500) and βactin (1:2000, #66009–1-Ig, ProteinTech, Chicago, IL, USA) were incubated with the membrane at 4 °C overnight.

Techniques: In Vivo, Over Expression, Knockdown, Immunohistochemical staining, Staining