Fig 1: Lnc-EGFR prevents the ubiquitination of EGFR by binding to Tyr1045.(a) T cells isolated from peripheral blood of HCC patients were transduced with indicated lentiviral particles and then treated with with EGF (20 ng ml-1) for indicated timepoints followed by western blotting for p-EGFR(Y1045), p-EGFR(Y1068), p-EGFR(Y1073), p-ERK1/2(T202/Y204), EGFR, ERK1/2 and ß-actin. (b) Normal, healthy human T cells transduced with mock or indicated lentiviral particles were determined with real-time PCR (upper) and were further treated with EGF (100 ng ml-1) for 90 min or left untreated. Whole-cell lysates were prepared and EGFR was immunoprecipitated followed by western blotting for ubiquitin. Equal loading of EGFR was determined by western blotting via anti-EGFR antibodies (lower). (c) Whole-cells lysates were prepared and c-CBL was immunoprecipitated via anti-c-CBL antibody. The presence of EGFR in the immunecomplex was determined by western blotting via anti-EGFR antibody. (d) Transduced T cells were treated with anti-CD3/anti-CD28 beads (bead-to-cell ratio of 1:1), EGF (20 ng ml-1) or left untreated in the presence or absence of PD98059 (40 µM) and/or CsA (1 µM). Whole-cell lysates were prepared and subjected to Western blotting for p-ERK1/2(T202/Y204), p-MEK1/2(S217/221), p-NF-AT1(S54), ERK1/2, IL-2, MEK1/2 and ß-actin. Each experiment was performed triplicated.
Fig 2: Overexpression of IDO affected the MAPK/ERK pathway. Western blot assay was performed on the protein expression levels of (A) p-Raf-1, Raf-1, p-Mek1/2 Mek1/2 (B) p-Erk1/2 and (C) Erk1/2 in 16HBE cells treated with control, NC, DEX, NC+DEX, and IDO+DEX. *P<0.05, **P<0.01 vs. control, ^P<0.05, ^^P<0.01 vs. NC. IDO, indoleamine 2, 3-dioxygenase; MAPK, mitogen-activated protein kinase; ERK, extracellular regulated kinase; p-Raf-1, phosphorylated-v-raf-1 murine leukemia viral oncogene homolog 1; p-Mek1/2, phosphorylated-mitogen-activated protein kinase; NC, negative control; DEX, dexamethasone.
Fig 3: Mechanism study of KIF15 KD in glioma. (A) Human apoptosis antibody array analysis was performed and analyzed in U87 MG cells with or without KIF15 KD. (B) The protein expression levels of Bax, p21 and Survivin in U251 cells were detected by western blot analysis. (C) The expression levels of Bax, p21 and Survivin were detected by IHC in tumor sections of a mouse xenograft model (magnification, ×200). (D) The expression levels of MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2 were detected by western blot analysis in U87 MG and U251 cells with or without KIF15 KD. *P<0.05, **P<0.01, ***P<0.001. KIF15, kinesin-12; KD, knockdown.
Fig 4: Syncytin-1 promotes doxorubicin resistance via MEK/ERK pathway in HCC.a CCK-8 assay. The effect of doxorubicin on cell viability was determined in HCCLM3 cells. b The regulation of doxorubicin on the expression of MEK/ERK pathway protein (MEK1/2, ERK1/2, p-MEK1/2, p-ERK1/2, CCND1, and CDK4) were detected by western blotting. c CCK-8 assay was used to investigate the role of Syncytin-1 on doxorubicin-induced cell apoptosis in Huh7 cells. d The function of Syncytin-1 overexpression on doxorubicin suppressed MEK/ERK pathway activation was assessed using western blotting. e Schema illustrates the mechanism by which Syncytin-1 promotes the development of HCC and doxorubicin resistance through activation of the MEK/ERK pathway. Syncytin-1 induced the activation of the MEK/ERK pathway and upregulation of its downstream proteins (such as c-myc, c-fos, c-jun, CCND1, and CDK4) levels in HCC. These genes participated in the malignant progression of HCC by promoting cell proliferation, migration, and invasion. Syncytin-1 also prevented HCC cells from doxorubicin-induced cell apoptosis. DOX, doxorubicin. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 5: Syncytin-1 activates MEK/ERK pathway in HCC.a The upregulated genes in HCC were analyzed by KEGG analysis. b and c The expression of p-MEK1/2 and p-ERK1/2 in 53 pairs of formalin-fixed HCC and adjacent tissues were detected using IHC. Representative images are shown (magnification, ×400). d The expression of p-MEK1/2 and p-ERK1/2 were determined using western blotting. The correlation between the expression of Syncytin-1 and p-MEK1/2 or p-ERK1/2 level was analyzed using Pearson’s correlation test. e The levels of p-MEK1/2, p-ERK1/2, and their downstream proteins (c-myc, c-fos, c-jun, CCND1, and CDK4) were examined in HCC cell lines transfected with Syncytin-1 or pSilencer-shSyncytin-1 by western blotting. f The levels of p-MEK1/2, p-ERK1/2, and their downstream proteins were detected after using MEK/ERK-specific inhibitors (JTP-74057, or GDC-0994) in Huh7 transfected with Syncytin-1. The bars represent results from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
Supplier Page from Abcam for Anti-MEK1 + MEK2 (phospho S218 + S222) antibody