rabbit anti eif4e total  (OriGene)

Journal: Frontiers in Physiology

Article Title: The Sphingolipid Inhibitors Ceranib-2 and SKI-II Reduce Measles Virus Replication in Primary Human Lymphocytes: Effects on mTORC1 Downstream Signaling

doi: 10.3389/fphys.2022.856143

Figure Lengend Snippet: Effects of ceranib-2 and SKI-II on rpS6, eIF4E, and MNK1 in PBL. In all experiments, the quality of the PBL after PHA (2.5 μg/ml) stimulation was controlled by flow cytometry measuring the expression of CD69 and CD25 (A) , and the infection with MV (MV IC323eGFP) measuring the expression virally encoded GFP (B) . An example of infected and uninfected PBL in the presence and absence of PHA is shown representatively. PBL were treated for indicated times with 5 μm ceranib-2, 5 μm SKI-II and as control with the solvent DMSO (0.5%), and cell lysates applied to SDS-PAGE and blotted on nitrocellulose. Proteins of interest were visualized on the Western blots using appropriate antibodies and quantified. The experiments were repeated with PBL from three different donors. In (C) , Western blotting was performed to detect phosphorylated rpS6 and GAPDH. The corresponding quantification of the blot is shown on the right side ( n = 3; blots from one representative experiment are shown). The reduction of rpS6 phosphorylation by ceranib-2 at 6 and 15 h was significant in comparison with corresponding time points of control signals (with * p ≤ 0.05 calculated by student’s t -test). In (D) , Western blotting was performed to detect phosphorylated eIF4E, total eIF4E, and GAPDH. The quantification with normalization to the corresponding DMSO control values for each time point is shown on the right side. The values for phosphorylated over total eIF4E signals are given. Significances were calculated by student’s t -test by comparing corresponding time points of inhibitor treated with control signals ( n = 3; with * p ≤ 0.05 and ** p ≤ 0.01). In (E) , Western blotting was performed to detect phosphorylated MNK1, total MNK1, and GAPDH. Quantifications of the phosphorylated over total MNK1 and total MNK1 protein over GAPDH are given. Some blots were used for detection of two different proteins (MNK1 and eIF4E) and GAPDH controls were used for both. In some cases, two blots with lysates from the same experiment were used for the detection of total and phosphorylated proteins, and the corresponding GAPDH controls were used for quantification. In this case, only one representative GAPDH control is shown.

Article Snippet: The following primary antibodies were used in immunoblotting and flow cytometry: rabbit anti-GAPDH (Santacruz sc-25,778), rabbit anti-human CD69 APC-conjugated (Biolegend 310,910) and FITC-conjugated (Biolegend 310,904), rabbit anti-human CD25 FITC-conjugated (Immunotools 218,102,535), rabbit anti-phosphoEIF4E (CST-9741), rabbit anti-total EIF4E (CST-9742), rabbit anti-phospho MNK1 (ThermoFischer PA5-110137), rabbit anti-total MNK1 (CST-2195), rabbit anti-total rpS6 (Sigma SAB4502676), rabbit anti-phospho rpS6 (CST-4858), rabbit anti-total IKK-β (CST-2684), and rabbit anti-mouse IgG1k FITC-conjugated (BD Biosciences 55,066).

Techniques: Flow Cytometry, Expressing, Infection, Control, Solvent, SDS Page, Western Blot, Phospho-proteomics, Comparison

Journal: Frontiers in Physiology

Article Title: The Sphingolipid Inhibitors Ceranib-2 and SKI-II Reduce Measles Virus Replication in Primary Human Lymphocytes: Effects on mTORC1 Downstream Signaling

doi: 10.3389/fphys.2022.856143

Figure Lengend Snippet: Effects of ceranib-2 and SKI-II on rpS6, eIF4E, MNK1, and IKK in MV-infected PBL. In (A) , Western blotting was performed to detect phosphorylated rpS6, total rpS6 protein, and GAPDH. The quantification with normalization to the corresponding DMSO control values for each time point is shown on the right side. The values for phosphorylated over total rpS6 and total rpS6 protein over GAPDH signals are given. The increase of total rpS6 in comparison with the DMSO control was significant at 16 and 24 h after infection. The 16 and 24 h values of ceranib-2-treated infected cells and the values of 6, 16, and 24 h SKI-II-treated infected cells were significantly reduced in comparison with both, uninfected and infected cell values. The phosphorylation of rpS6 was significantly reduced by SKI-II at 16 and 24 h in comparison with infected cell values ( n = 3; with * p ≤ 0.05). In (B) , Western blotting was performed to detect phosphorylated eIF4E, total eIF4E protein, and GAPDH. The quantification with normalization to the corresponding DMSO control values for each time point is shown on the right side. The values for phosphorylated over total eIF4E signals are given. Significances were calculated by student’s t -test by comparing corresponding time points of inhibitor treated with control signals ( n = 3; with * p ≤ 0.05). In (C) , Western blotting was performed to detect phosphorylated MNK1, total MNK1 protein, and GAPDH. Only the values for total MNK1 over GAPDH signals are given, because there were no significant differences in the phosphorylation of MNK1. Significances were calculated by student’s t -test by comparing corresponding time points of inhibitor treated with control signals ( n = 3; with * p ≤ 0.05). In (D) , Western blotting was performed to detect total IKK protein and GAPDH. The quantification with normalization to the corresponding DMSO control values for each time point is shown on the right side. The values for total IKK over GAPDH signals are given. Significances were calculated by student’s t -test by comparing corresponding time points of inhibitor treated with control signals ( n = 3; with * p ≤ 0.05). Some blots were used for detection of two different proteins (MNK1 and IKK) and GAPDH controls were re-used, and in some cases, two blots with lysates from the same experiment were used for the detection of total and phosphorylated proteins. The corresponding GAPDH controls were used for quantification. Only blots from one representative experiment are shown.

Article Snippet: The following primary antibodies were used in immunoblotting and flow cytometry: rabbit anti-GAPDH (Santacruz sc-25,778), rabbit anti-human CD69 APC-conjugated (Biolegend 310,910) and FITC-conjugated (Biolegend 310,904), rabbit anti-human CD25 FITC-conjugated (Immunotools 218,102,535), rabbit anti-phosphoEIF4E (CST-9741), rabbit anti-total EIF4E (CST-9742), rabbit anti-phospho MNK1 (ThermoFischer PA5-110137), rabbit anti-total MNK1 (CST-2195), rabbit anti-total rpS6 (Sigma SAB4502676), rabbit anti-phospho rpS6 (CST-4858), rabbit anti-total IKK-β (CST-2684), and rabbit anti-mouse IgG1k FITC-conjugated (BD Biosciences 55,066).

Techniques: Infection, Western Blot, Control, Comparison, Phospho-proteomics

Journal: Frontiers in Physiology

Article Title: The Sphingolipid Inhibitors Ceranib-2 and SKI-II Reduce Measles Virus Replication in Primary Human Lymphocytes: Effects on mTORC1 Downstream Signaling

doi: 10.3389/fphys.2022.856143

Figure Lengend Snippet: Schematic summary of the major findings. (A) Schematically summarizes the observed effects of ceranib-2 and SKI-II in uninfected PBL, (B) shows the effect of MV infection on these signaling pathways, and (C) the effects of the two inhibitors on infected PBL. Whereas in uninfected cells, the major effect was found on the phosphorylation of eIF4E, in MV-infected cells ceranib-2 and SKI-II predominantly neutralize the effect of the infection and reduce rpS6 expression below levels in untreated control cells.

Article Snippet: The following primary antibodies were used in immunoblotting and flow cytometry: rabbit anti-GAPDH (Santacruz sc-25,778), rabbit anti-human CD69 APC-conjugated (Biolegend 310,910) and FITC-conjugated (Biolegend 310,904), rabbit anti-human CD25 FITC-conjugated (Immunotools 218,102,535), rabbit anti-phosphoEIF4E (CST-9741), rabbit anti-total EIF4E (CST-9742), rabbit anti-phospho MNK1 (ThermoFischer PA5-110137), rabbit anti-total MNK1 (CST-2195), rabbit anti-total rpS6 (Sigma SAB4502676), rabbit anti-phospho rpS6 (CST-4858), rabbit anti-total IKK-β (CST-2684), and rabbit anti-mouse IgG1k FITC-conjugated (BD Biosciences 55,066).

Techniques: Infection, Protein-Protein interactions, Phospho-proteomics, Expressing, Control

Journal: Molecular human reproduction

Article Title: The effect of heat shock protein 27 on extravillous trophoblast differentiation and on eukaryotic translation initiation factor 4E expression.

doi: 10.1093/molehr/gau002

Figure Lengend Snippet: Figure 3 Ribavirin elevates HSP27 and eIF4E levels in placental explants. In order to further establish the connection between HSP27, cell differentiation and eIF4E expression, we exposed first tri- mester human placental explants to ribavirin (10 mM, 72 h) that facili- tates EVT cell differentiation. Its effects on HSP27 and eIF4E expression were analyzed using western blot. *Significantly different from control siRNA (P , 0.05, n ≥4).

Article Snippet: The number of sectors in which EVT cells appeared was ............................................................................................................................................................................................. Target Source Isotype Company Dilutions Method Human HLA-G Mouse mAb IgG1 Acris 1:500 WB 1:200 IHC Human HSP27 Mouse mAb IgG1 Chemicon 1:200 WB 1:100 IHC Human Ki67 Mouse mAb IgG1 Zymed 1:150 IHC Human Caspase 3 (cleaved) Rabbit Polyclonal IgG Biocare medical 1:50 IHC Human E-cadherin Rabbit mAb IgG Cell Signaling Technology 1:1000 WB IgG Isotype control Rabbit Polyclonal IgG Dako 1:20000 IHC IgG1 Isotype control Mouse mAb IgG1 R&D Systems 1:2000 IHC Human total eIF4E Rabbit Polyclonal IgG Cell Signaling Technology 1:1000 WB 1:100 IHC Human Phospho-eIF4E (Ser209) Rabbit Polyclonal IgG Cell Signaling Technology 1:1000 WB Human total 4E-BP1 Rabbit Polyclonal IgG Cell Signaling Technology 1:1000 WB Human Phospho-4EBP1 (ser65) Rabbit mAb IgG Cell Signaling Technology 1:1000 WB Human total MNK1 Rabbit mAb IgG Cell Signaling Technology 1:1000 WB Human phospho-MNK1 (Thr197/202) Rabbit mAb IgG Epitomics 1:1000 WB Human Phospho-P53 (Ser 46) Rabbit Polyclonal IgG Cell Signaling Technology 1:1000 WB Human Cyclin D1 Rabbit Polyclonal IgG Cell Signaling Technology 1:1000 WB Human CD9 Mouse mAb IgG1 Acris 1:500 WB Human Tubulin Mouse mAb IgG1 Sigma 1:8000 WB Peroxidase conjugated anti-mouse Goat Polyclonal IgG Jackson Immuno-Research 1:40000 WB Peroxidase conjugated anti-Rabbit Goat Polyclonal IgG Jackson Immuno-Research 1:10000 WB SuperPicTure Polymer detection kit (HRP) Zymed Lab IHC D ow nloaded from https://academ ic.oup.com /m olehr/article/20/5/422/1214684 by Indian Institute of Technology Bom bay user on 16 June 2024 calculated [for example, nine sectors were considered as 75% effect (9/12 × 100 1⁄4 75%)].

Techniques: Cell Differentiation, Expressing, Western Blot, Control

Journal: Molecular human reproduction

Article Title: The effect of heat shock protein 27 on extravillous trophoblast differentiation and on eukaryotic translation initiation factor 4E expression.

doi: 10.1093/molehr/gau002

Figure Lengend Snippet: Figure 5 The effect of HSP27 silencing on eIF4E expression and its regulators. In order to further establish the connection between HSP27 and eIF4E ex- pression, we analyzed the effect of HSP27 silencing on total and phosphorylation levels of eIF4E and its regulators MNK1 and 4EBP1 using western blot. (A) Representative photomicrographs of paraffin sections of placental explants exposed to control/HSP27 siRNA stained for eIF4E. (B) Western blot analyses of eIF4E expression levels of placental explants exposed to control/HSP27 siRNA. (C) Western blot analyses of total and phosphorylated expression levels of eIF4E, 4EBP1 and MNK1 of HTR8/SVneo cells exposed to control/HSP27 siRNA. *Significantly different from control siRNA (P , 0.05, n ≥4).

Article Snippet: The number of sectors in which EVT cells appeared was ............................................................................................................................................................................................. Target Source Isotype Company Dilutions Method Human HLA-G Mouse mAb IgG1 Acris 1:500 WB 1:200 IHC Human HSP27 Mouse mAb IgG1 Chemicon 1:200 WB 1:100 IHC Human Ki67 Mouse mAb IgG1 Zymed 1:150 IHC Human Caspase 3 (cleaved) Rabbit Polyclonal IgG Biocare medical 1:50 IHC Human E-cadherin Rabbit mAb IgG Cell Signaling Technology 1:1000 WB IgG Isotype control Rabbit Polyclonal IgG Dako 1:20000 IHC IgG1 Isotype control Mouse mAb IgG1 R&D Systems 1:2000 IHC Human total eIF4E Rabbit Polyclonal IgG Cell Signaling Technology 1:1000 WB 1:100 IHC Human Phospho-eIF4E (Ser209) Rabbit Polyclonal IgG Cell Signaling Technology 1:1000 WB Human total 4E-BP1 Rabbit Polyclonal IgG Cell Signaling Technology 1:1000 WB Human Phospho-4EBP1 (ser65) Rabbit mAb IgG Cell Signaling Technology 1:1000 WB Human total MNK1 Rabbit mAb IgG Cell Signaling Technology 1:1000 WB Human phospho-MNK1 (Thr197/202) Rabbit mAb IgG Epitomics 1:1000 WB Human Phospho-P53 (Ser 46) Rabbit Polyclonal IgG Cell Signaling Technology 1:1000 WB Human Cyclin D1 Rabbit Polyclonal IgG Cell Signaling Technology 1:1000 WB Human CD9 Mouse mAb IgG1 Acris 1:500 WB Human Tubulin Mouse mAb IgG1 Sigma 1:8000 WB Peroxidase conjugated anti-mouse Goat Polyclonal IgG Jackson Immuno-Research 1:40000 WB Peroxidase conjugated anti-Rabbit Goat Polyclonal IgG Jackson Immuno-Research 1:10000 WB SuperPicTure Polymer detection kit (HRP) Zymed Lab IHC D ow nloaded from https://academ ic.oup.com /m olehr/article/20/5/422/1214684 by Indian Institute of Technology Bom bay user on 16 June 2024 calculated [for example, nine sectors were considered as 75% effect (9/12 × 100 1⁄4 75%)].

Techniques: Expressing, Phospho-proteomics, Western Blot, Control, Staining