antibodies against p-eif4e  (Cell Signaling Technology Inc)

Journal: Cell Death & Disease

Article Title: CXCR7 promotes melanoma tumorigenesis via Src kinase signaling

doi: 10.1038/s41419-019-1442-3

Figure Lengend Snippet: a The levels of Src phosphorylation in F10 WT cells transfected with siRNA targeting scramble control (NC), β-arrestin1 (βArr1), or β-arrestin2 (βArr2). b , e The effects of β-arrestin2 knockdown on CXCL12-induced Src phosphorylation in melanoma cells. F10 WT ( b ) and A375 WT ( e ) cells harboring scramble siRNA or β-arrestin2 siRNA were starved overnight. After pretreated with AMD3100 (1 μg/ml) for 1 h, the cells were exposed to recombinant murine (50 ng/ml) or human (100 ng/ml) CXCL12. The phosphorylation level of Src was detected by western blot. c , f The effects of β-arrestin2 downregulation on melanoma cell proliferation. Indicated cell lines were transfected with scramble siRNA or β-arrestin2 siRNA and seeded into 96-well plates. After 48 h, the cell numbers were determined by CCK-8 assays. The proliferation rates were normalized to F10 WT cells ( c , top), F0 Vec cells ( c bottom) or A375 WT cells ( f ) transfected with scramble siRNA. d , g The effects of β-arrestin2 downregulation on melanoma colony formation. Indicated cell lines were transfected with scramble siRNA or β-arrestin2 siRNA and seeded into 6-well plates. After 12 days, the colony numbers were counted. The quantitative results were normalized to F10 WT cells ( d , top), F0 Vec cells ( d , bottom) or A375 WT cells ( g ) transfected with scramble siRNA. Proliferation experiments and colony formation assays were independently repeated in triplicate. Data are presented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001; ns not significant

Article Snippet: Antibodies against p-AKT, AKT, p-Src, Src, p-S6K, S6K, p-ERK, ERK, p-4E-BP1, 4E-BP1, p-eIF4E, eIF4E, and β-arrestin2 were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Transfection, Control, Knockdown, Recombinant, Western Blot, CCK-8 Assay

Journal: Cell Death & Disease

Article Title: CXCR7 promotes melanoma tumorigenesis via Src kinase signaling

doi: 10.1038/s41419-019-1442-3

Figure Lengend Snippet: a HIF-1α expression in murine melanoma cells treated with MG132 (10 μM). b The effects of CXCR7 modifications on eIF4E phosphorylation in melanoma cells. c , d CXCL12 stimulated eIF4E phosphorylation through the Src signaling in melanoma cells. F10 WT ( c ) and A375 WT ( d ) cells were starved overnight. After pretreated with AMD3100 (1 μg/ml) combined with PP1 (10 μM) or U0126 (10 μM) for 1 h, the cells were stimulated with recombinant murine (50 ng/ml) or human (100 ng/ml) CXCL12 as indicated. The cell lysates were collected and immunoblotted with the indicated antibodies. e A375 WT and KO cells were pretreated with cercosporamide (cerco, 20 μM) overnight and then incubated with CoCl 2 (200 μM) for 4 h. The levels of HIF-1α and phosphorylated eIF4E were examined by western blot. f The proposed model for the functions of CXCR7 in melanoma tumorigenesis. CXCR7 is activated by CXCL12 to support melanoma cell proliferation through β-arrestin2-mediated Src phosphorylation. In addition, the activation of this CXCR7-Src axis stimulates eIF4E phosphorylation to accelerate the translation of HIF-1α, which enhances the secretion of VEGF to facilitate melanoma angiogenesis

Article Snippet: Antibodies against p-AKT, AKT, p-Src, Src, p-S6K, S6K, p-ERK, ERK, p-4E-BP1, 4E-BP1, p-eIF4E, eIF4E, and β-arrestin2 were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Phospho-proteomics, Recombinant, Incubation, Western Blot, Activation Assay

Journal: Oncotarget

Article Title: Metformin requires 4E-BPs to induce apoptosis and repress translation of Mcl-1 in hepatocellular carcinoma cells

doi: 10.18632/oncotarget.10671

Figure Lengend Snippet: A . Western blot of indicated proteins in control (scrambled) and 4E-BP1/2 KD Huh7 cells treated with 10 mM metformin for 12, 24, or 48 hours. β-actin was used as a loading control; B . m 7 GDP pull-down assay. WT and 4E-BP1/2 KD Huh7 cells were treated with 10 mM metformin for the indicated time. Cells were lysed and cell lysates were incubated with m 7 GDP-agarose for 2 hours at 4°C. eIF4E, eIF4G and 4E-BP1 are shown by western blotting; C . Metformin induces apoptosis to a higher extent in scrambled versus 4E-BP1/2-depleted cells, as illustrated by a stronger increase in the percentage of apoptotic nuclei by DAPI assay (p=0.012), and caspase 3/7 positivity (p=0.0039) at 48 hours; D . Polysome profiles of scrambled (control) and 4E-BP1/2 KD Huh7 cells treated with vehicle or 10 mM metformin for 12 hours. Absorbance at 254 nm was continuously recorded; E . Distribution of Mcl-1 and β-actin mRNAs fractionated across the density gradients from (D) was determined by RT-qPCR; F . Expression of total Mcl-1 and β-actin mRNAs in cells treated as described in (D) was determined by RT-qPCR.

Article Snippet: Antibody against eIF4E was purchased from Novus Biologicals (Littleton, CO).

Techniques: Western Blot, Control, Pull Down Assay, Incubation, Quantitative RT-PCR, Expressing

Journal: Oncotarget

Article Title: Metformin requires 4E-BPs to induce apoptosis and repress translation of Mcl-1 in hepatocellular carcinoma cells

doi: 10.18632/oncotarget.10671

Figure Lengend Snippet: A . and B . A patient microarray of 60 independent tumor tissues show a high eIF4E/4E-BP1 ratio in 33.0% of tumors, an equivalent eIF4E/4E-BP1 ratio in 60.0% of tumors, and a low eIF4E/4E-BP1 ratio in 7.0% of tumors. Kaplan-Meier curve demonstrates that tumors with a high eIF4E/4E-BP1 ratio are associated with decreased survival as compared to tumors with a low eIF4E/4E-BP1 ratio; C . Clinical and demographic characteristics of patients with Hepatocellular carcinoma tumors represented on tissue microarray, according to eIF4E/4E-BP1 ratio measured by immunohistochemistry; D . Proposed model for metformin induction of apoptosis via 4E-BPs. In cells with low to equivalent eIF4E/4E-BP1 ratio, metformin represses Mcl-1 mRNA translation and induces apoptosis of liver cancer cells via activation of 4E-BPs, whereas in tumors with low or absence of 4E-BP1/2 expression (or potentially higher expression of eIF4E), Mcl-1 mRNA translation is sustained and cells are resistant to metformin-induced apoptosis.

Article Snippet: Antibody against eIF4E was purchased from Novus Biologicals (Littleton, CO).

Techniques: Microarray, Immunohistochemistry, Activation Assay, Expressing

Journal: Molecular cancer research : MCR

Article Title: Differential Regulation of ZEB1 and EMT by MAPK-interacting protein kinases (MNKs) and eIF4E in Pancreatic Cancer

doi: 10.1158/1541-7786.MCR-15-0285

Figure Lengend Snippet: A, PDAC cells (AsPC1, CD18 and Panc1) were grown on tissue culture plastic or in three-dimensional type I collagen (2mg/ml) for 24 hours. The cell lysates were analyzed for eIF4E phosphorylation on Ser209 (p-eIF4E) by Western blotting. B, PDAC cells were grown for 24 hours on tissue culture plastic or in three-dimensional type I collagen (2mg/ml). The cell lysates were analyzed for MNK1 phosphorylation on Thr197/202 by Western blotting. C, PDAC cells growing in three-dimensional type I collagen were treated with DMSO or CGP57380 (2.5 μM) for 24 hours and the effect on eIF4E phosphorylation was determined by Western blotting. D, PDAC cells were transfected with control siRNA (siCtrl) or a combination of MNK1 and MNK2 specific siRNAs (siMNK1/2) for 48 hours. The cells were then grown in three-dimensional type I collagen (2mg/ml) for additional 24 hours. The effect on MNK1 and MNK2 mRNA expression was determined by qRT-PCR. Effect on MNK1 protein levels and eIF4E phosphorylation was determined by Western blotting. The results are representative of at least three independent experiments. E, Human pancreatic TMAs containing 27 pancreatic tumor specimens were immunostained with anti-p-eIF4E antibody and assessed for fibrosis using trichrome staining. Samples were obtained on an IRB-approved protocol. The relative immunostaining and the extent of fibrosis were graded as low (0 or 1+) or high (2+ or 3+). The relationship between p-eIF4E expression in the tissue samples and the extent of fibrosis was assessed by Fisher’s exact test.

Article Snippet: Antibodies against eIF4E, tubulin, HSP90, ZEB1 and Dicer were obtained from Santa Cruz, while antibodies against p-eIF4E, MNK1, p-MNK1 and Drosha were purchased from Cell Signaling.

Techniques: Phospho-proteomics, Western Blot, Transfection, Control, Expressing, Quantitative RT-PCR, Staining, Immunostaining

Journal: Molecular cancer research : MCR

Article Title: Differential Regulation of ZEB1 and EMT by MAPK-interacting protein kinases (MNKs) and eIF4E in Pancreatic Cancer

doi: 10.1158/1541-7786.MCR-15-0285

Figure Lengend Snippet: Chemoresistant CD18 (CD18-CR) cells were generated as detailed in Materials and Methods. A, CD18 and CD8-CR cells growing on tissue culture plastic were examined by phase contrast microscopy. Lysates from CD18 and CD18-CR cells were analyzed for E-cadherin, vimentin and ZEB1 protein and mRNA expression by Western blotting and qRT-PCR. B, CD18 and CD18-CR cells were plated onto thin-layer type I collagen matrix overlaid with colloidal gold, allowed to migrate for 24 hours, and the tracks were photographed and quantified. *, p<0.05. C, CD18 and CD18-CR cells were grown on tissue culture plastic or in three-dimensional type I collagen (2mg/ml) for 24 hours. The cell lysates were analyzed for eIF4E phosphorylation on Ser209 by Western blotting. D, CD18-CR cells growing in three-dimensional type I collagen were treated with DMSO or CGP57380 (2.5 μM) for 24 hours and the effect on eIF4E phosphorylation was determined by Western blotting. E, CD18-CR cells were transfected with control siRNA (siCtrl) or a combination of MNK1 and MNK2 specific siRNAs for 48 hours. The cells were then grown in three-dimensional type I collagen (2mg/ml) for additional 24 hours. The effect on MNK1 and MNK2 mRNA expression was determined by qRT-PCR. Effect on MNK1 protein levels and eIF4E phosphorylation was determined by Western blotting. The results are representative of at least three independent experiments. See also Supplemental Figure S1.

Article Snippet: Antibodies against eIF4E, tubulin, HSP90, ZEB1 and Dicer were obtained from Santa Cruz, while antibodies against p-eIF4E, MNK1, p-MNK1 and Drosha were purchased from Cell Signaling.

Techniques: Generated, Microscopy, Expressing, Western Blot, Quantitative RT-PCR, Phospho-proteomics, Transfection, Control

Journal: Molecular cancer research : MCR

Article Title: Differential Regulation of ZEB1 and EMT by MAPK-interacting protein kinases (MNKs) and eIF4E in Pancreatic Cancer

doi: 10.1158/1541-7786.MCR-15-0285

Figure Lengend Snippet: A, CD18-CR cells were treated with CGP57380 (2.5 μM) for 72 hours and the effect on E-cadherin and vimentin protein was determined by Western blotting (left). The effect on E-cadherin and vimentin mRNA expression was determined by qRT-PCR (right). B, CD18-CR previously treated with DMSO or CGP57380 (2.5 μM) for 48 hours were plated onto thin-layer type I collagen matrix overlaid with colloidal gold, allowed to migrate for 24 hours in the presence of DMSO or CGP57380 (2.5 μM), and the tracks were photographed and quantified. *, p<0.05. C and D, CD18-CR cells were transfected with control siRNA (siCtrl), MNK1 siRNA (siMNK1), MNK2 siRNA (siMNK2) or a combination of MNK1 and MNK2 specific siRNAs (siMNK1/2) for 48 hours. The cells were then grown in three-dimensional type I collagen (2mg/ml) for additional 24 hours. The effect on p-eIF4E, E-cadherin and vimentin expression was determined by Western blotting (C). The effect on E-cadherin and vimentin mRNA expression was determined by qRT-PCR (D). The results are representative of at least three independent experiments.

Article Snippet: Antibodies against eIF4E, tubulin, HSP90, ZEB1 and Dicer were obtained from Santa Cruz, while antibodies against p-eIF4E, MNK1, p-MNK1 and Drosha were purchased from Cell Signaling.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Transfection, Control

Journal: Molecular cancer research : MCR

Article Title: Differential Regulation of ZEB1 and EMT by MAPK-interacting protein kinases (MNKs) and eIF4E in Pancreatic Cancer

doi: 10.1158/1541-7786.MCR-15-0285

Figure Lengend Snippet: A, CD18-CR cells were transfected with control siRNA (siCtrl) or eIF4E-specific siRNA (sieIF4E) for 48 hours. The cells were then grown in three-dimensional type I collagen (2mg/ml) for additional 24 hours. The cells were processed for eIF4E, ZEB1, E-cadherin and vimentin mRNA expression by qRT-PCR and the lysates were analyzed for eIF4E, ZEB1, E-cadherin and vimentin protein expression by Western blotting. CD18-CR cells growing on glass coverslips were transfected with siCtrl, sieIF4E or a combination of MNK1 and MNK2 specific siRNAs (siMNK1/2) for 72 hours. The cells were then stained for actin using phalloidin and DAPI to counterstain the nuclei. B, CD18-CR cells were transfected with siCtrl or sieIF4E for 48 hours. The cells were then grown in three-dimensional type I collagen (2mg/ml) for additional 24 hours. The cells were processed for miR-200b, miR-200a, miR200c and miR-141 microRNAs and normalized using RNU48 as internal control. C, CD18-CR cells were transfected with siCtrl or siMNK1/2 for 48 hours. The cells were then grown in three-dimensional type I collagen (2mg/ml) for additional 24 hours. The cells were then processed for miR-200b, miR-200a, miR200c and miR-141 microRNAs and normalized using RNU48 as internal control. D, CD18-CR cells were transfected with control siRNA (siCtrl) or hnRNPA1-specific siRNA (sihnRNPA1) for 48 hours. The cells were then grown in three-dimensional type I collagen (2mg/ml) for additional 24 hours. The cells were processed for hnRNPA1, ZEB1, E-cadherin and vimentin mRNA expression by qRT-PCR and the lysates were analyzed for hnRNPA1, ZEB1, E-cadherin and vimentin protein expression by Western blotting. The results are representative of three independent experiments. See also Supplemental Figures S2–5.

Article Snippet: Antibodies against eIF4E, tubulin, HSP90, ZEB1 and Dicer were obtained from Santa Cruz, while antibodies against p-eIF4E, MNK1, p-MNK1 and Drosha were purchased from Cell Signaling.

Techniques: Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Staining

Journal: Molecular cancer research : MCR

Article Title: Differential Regulation of ZEB1 and EMT by MAPK-interacting protein kinases (MNKs) and eIF4E in Pancreatic Cancer

doi: 10.1158/1541-7786.MCR-15-0285

Figure Lengend Snippet: A, Pancreatic cancer organoids were established from a de-identified human PDAC tumor specimen using the published Tuveson Lab protocol as detailed in Materials and Methods. B, Three different pancreatic cancer organoids were treated with DMSO or CGP57380 (2.5 μM) for 96 hours and the effects on ZEB1, E-cadherin, and vimentin mRNA levels were determined by qRT-PCR. n.d., not detected. The p values were calculated using paired t test. C, Pancreatic cancer organoids were treated with DMSO or CGP57380 (2.5 μM) for 7 days, and the effect on pancreatic organoid size was examined by phase-contrast microscopy and size of the individual organoids was measured. Shown here is box plot with whiskers representing 10th-90th percentiles. ***, p<0.001. D, The model depicts differential regulation of ZEB1 by MNKs and eIF4E: Targeting MNKs attenuates EMT by inhibiting the translation of ZEB1 in pancreatic cancer cells, while downregulating the MNK effector eIF4E increases ZEB1 through repression of ZEB1-targeting microRNAs miR-200c and miR-141. Note that while we demonstrate that hnRNPA1 functions as a translational repressor of ZEB1 mRNA in PDAC cells, Buxade et al. have previously shown that MNK-mediated phosphorylation of hnRNPA1 relieves its repression of mRNA translation (15).

Article Snippet: Antibodies against eIF4E, tubulin, HSP90, ZEB1 and Dicer were obtained from Santa Cruz, while antibodies against p-eIF4E, MNK1, p-MNK1 and Drosha were purchased from Cell Signaling.

Techniques: Quantitative RT-PCR, Microscopy, Phospho-proteomics