Fig. 3: Shoc2-dependent activation of the ERK1/2 pathway in endothelial cells.
From: Signaling scaffold Shoc2 regulates lymphangiogenesis by suppressing mTORC1-mediated IFN responses
A Western blot of whole cell lysates extracted from human dermal lymphatic endothelial cells (HDLEC) expressing lentivirally supplied non-targeting shRNA (NT), Shoc2 shRNA (KD), or Shoc2 shRNA together with Shoc2-tagRFP (SE), serum-starved for 16 h and then stimulated or not with VEGF-C (2 ng/ml) for 7 min at 37 °C. Cell lysates were probed using anti-pERK1/2, -ERK1/2, and -Shoc2 antibodies. Shoc2-tR indicates Shoc2-tagRFP with a higher molecular weight. B Levels of pERK1/2 in HDLEC treated with 2 ng/ml VEGFa at 7 min, normalized to the total amount of ERK1/2 and compared to pERK1/2 levels in HDLEC expressing non-targeting (NT) treated with 2 ng/ml VEGFa (set at 1.0). Values are shown in arbitrary units ± S.D. (n = 3) (*p = 0.01 to 0.05, **p = 0.001 to 0.01, ***p = 0.0001 to 0.001, ****p < 0.0001, ≥ 0.05- non-significant, ns, one-way ANOVA Dunnett’s multiple comparison test). C Western blot of whole cell lysates extracted from human umbilical vein endothelial cells (HUVEC) expressing lentivirally supplied non-targeting shRNA (NT), Shoc2 shRNA (KD) or Shoc2 shRNA together with Shoc2-tagRFP (SE), serum-starved for 16 h and then stimulated or not with VEGF-A (2 ng/ml) for 7 min at 37 °C. Cell lysates were probed using anti-pERK1/2, -ERK1/2, and -Shoc2 antibodies. D Levels of pERK1/2 in HUVEC treated with 2 ng/ml VEGFa at 7 min, normalized to the total amount of ERK1/2 and compared to pERK1/2 levels in HUVEC cells expressing non-targeting (NT), treated with 2 ng/ml VEGFa (set at 1.0). Values are shown in arbitrary units ± S.D. (n = 3) (*p = 0.01 to 0.05, **p = 0.001 to 0.01, ***p = 0.0001 to 0.001, ****p < 0.0001, ≥ 0.05- non-significant, ns, one-way ANOVA Dunnett’s multiple comparison test). E HDLEC expressing lentivirally-supplied non-targeting shRNA (NT) or Shoc2 shRNA (KD) were stained to examine β-Galactosidase activity (left panels). GFP, expressed independently by the lentivirus, served as a reporter to monitor delivery efficiency (right panels). Scale bar–150 μm. F Size distribution of HDLEC expressing lentivirally-supplied non-targeting shRNA (NT) or Shoc2 shRNA (KD), 6 days after transfection, as measured by surface area. G Western blot of whole cell lysates extracted from HDLECs and HUVECs expressing lentivirally supplied non-targeting shRNA (NT) or Shoc2 shRNA (KD) for 6 days. Cell lysates were probed using anti-Shoc2, -TP53, -pTP53, -CDKNA1, -CDKN2A, -IL-6, -pH2A.X and -GAPDH antibodies. The results in each panel are representative of those from three independent experiments.
