Fig 1: (a–b) Effects of LPS on CXCR4, 7 protein levels in u‐THP‐1 and d‐THP‐1. Cells were cultured and treated with or without LPS. Cells were harvested for protein determination and western blot as described in Materials and Methods. Immunoreactive bands were quantitated using ODYSSEY® CLx Infrared Imaging System as described in Materials and Methods. Results were normalized to vehicle control and expressed as relative protein levels (mean ± SD, n = 3). p values ≤ 0.05 were considered significant and * indicates significantly different from control. The panel represents (a) β‐actin and CXCR7. (b) β‐actin and CXCR4.
Fig 2: Change profile of downstream functional proteins after the inhibition of STAT1 activity. (A) Western blotting revealed that, after knocking down the expression of JAK2 by siRNA transfection and repressing the activity of JAK2 by FLUD in C4-2B cells, the expression of CXCR7 and P-JAK2 did not display any change, but the expression of C-Myc and Bcl-2 decreased remarkably. In contrast, the expression of DNA damage marker γH2AX increased apparently, the expression of homologous repairing (HR) protein C-PARP-1 increased accordingly, but the expression of non-homologous end-joining (NHEJ) repairing protein DNA-PKCs decreased measurably. (B) All WB band densities were quantitative analyzed.
Fig 3: (a–g) Effects of anti‐CD14 antibody on LPS induction of gene expression in d‐THP‐1 cells. d‐THP‐1 were treated with mouse IgG isotype control or anti‐CD14 antibody (10 μg/mL) for one hour then medium was replaced with fresh medium with or without 10 ng/mL of LPS for an additional four hours. After LPS treatment, cells were harvested, total RNA isolated and mRNA levels of CXCL12, CXCR4, CXCR7, TNF‐α, TLR4, IL‐1β, IL‐6, and CCL2 were determined using RT‐PCR as described in Materials and Methods. Results were normalized to vehicle control and expressed as relative mRNA levels (mean ± SD, n = 3). p values ≤ 0.05 were considered significant and * indicates significantly different from control. (a) CXCR12. (b) CXCR4. (c) CXCR7. (d) TNF‐α. (e) TLR4 (f) IL‐1β. (g) IL‐6. (h) CCL2.
Fig 4: Change profile of downstream functional proteins after the inhibition of JAK2 activity. (A) Western blotting revealed that, after knocking down the expression of JAK2 by siRNA transfection and repressing the activity of JAK2 by AZD1480 in C4-2B cells, the expression of CXCR7 did not display any change, but the expression of p-STAT1, oncogene C-Myc, and anti-apoptotic protein Bcl-2 decreased significantly. In addition, the expression of DNA damage marker γH2AX increased, the expression of non-homologous end-joining (NHEJ) repairing protein DNA-PKCs significantly decreased, but the expression of homologous repairing (HR) protein C-PARP-1 increased. (B) All WB band densities were quantitatively analyzed.
Fig 5: Comparison of CXCR7 downstream signaling proteins between CCX771-based treatment and ENZ-based treatment. (A) Western blotting demonstrated that the expression of phosphorylated JAK2, phosphorylated STAT1, C-Myc, and VEGFR markedly decreased, but the expression of cleaved-PARP-1 increased in CCX771-based treatment cells. The combination treatment of CCX771 + ENZ exhibited much greater synergistic suppression of phosphorylated JAK2, phosphorylated STAT1, C-Myc, and VEGFR. (B) All western blot band densities were quantitatively analyzed.
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