Journal:

Article Title: Mnk2 and Mnk1 Are Essential for Constitutive and Inducible Phosphorylation of Eukaryotic Initiation Factor 4E but Not for Cell Growth or Development

doi: 10.1128/MCB.24.15.6539-6549.2004

Figure Lengend Snippet: Phosphorylation of eIF4E by Mnk1 and Mnk2 in mouse embryonic fibroblasts. (A) Fibroblasts prepared from WT or DKO embryos were serum starved for 20 h and either left unstimulated (−) or stimulated with FCS (25%, 15 min), TPA (500 nM, 15 min), anisomycin (10 μg/ml, 15 min), UV-C irradiation (40 J/m2, then incubated for 20 min), TNF-α (10 ng/ml, 30 min), IL-1β (10 ng/ml, 15 min), or osmotic shock (additional 0.2 M NaCl, 30 min) followed by isotonic recovery (30 min). Cells growing exponentially in DMEM containing 10% FCS were prepared in parallel (growing). Cell lysates (10 μg protein) were analyzed by immunoblotting with anti-phospho-Mnk1 (Thr197/202), anti-phospho-eIF4E (Ser209), anti-eIF4E, anti-phospho-ERK, anti-phospho-p38, and anti-phospho-HSP27/25 antibodies, as indicated on to the right of the panel. The positions of the phospho-Mnk1 and -Mnk2 proteins are indicated by filled and open arrowheads, respectively. (B) Embryonic fibroblasts of the indicated genotypes were treated as described for panel A, and cell lysates (10 μg of protein) were analyzed by immunoblotting with anti-phospho-eIF4E (Ser209), anti-eIF4E, anti-Mnk1, and anti-Mnk2 antibodies. The asterisks indicate nonspecific signals (ns). (C) Embryonic fibroblasts from WT, Mnk1 KO (1-KO), Mnk2 KO (2-KO), or DKO mice were radiolabeled with [32P]orthophosphate under serum-starved conditions for 20 h and then stimulated with 500 nM TPA for 15 min. eIF4E was purified from cell extracts by either m7GTP-Sepharose affinity resin or immunoprecipitation with an anti-eIF4E monoclonal antibody. Samples were resolved by SDS-PAGE, electrotransferred to a PVDF membrane, and exposed to an X-ray film for autoradiography (upper panel). The filter was then subjected to immunoblotting with the anti-eIF4E antibody (lower panel). IgL, immunoglobulin light chain; circled P, phospho.

Article Snippet: For the immunopurification of eIF4E, 1 μg of an anti-eIF4E monoclonal antibody (clone P-2; Santa Cruz) and 20 μl of protein G-Sepharose were added to 0.4 ml of the cell extract and incubated for 1 h at 4°C.

Techniques: Irradiation, Incubation, Western Blot, Purification, Immunoprecipitation, SDS Page, Autoradiography

Journal:

Article Title: Mnk2 and Mnk1 Are Essential for Constitutive and Inducible Phosphorylation of Eukaryotic Initiation Factor 4E but Not for Cell Growth or Development

doi: 10.1128/MCB.24.15.6539-6549.2004

Figure Lengend Snippet: Phosphorylation of eIF4E by Mnk1 and Mnk2 in mouse tissues. (A) Tissue extracts (18 μg of protein) prepared from WT, Mnk1 KO (1K), Mnk2 KO (2K), and DKO (DK) mice were analyzed by immunoblotting with anti-Mnk1, anti-Mnk2, anti-phospho-eIF4E (Ser209), and anti-eIF4E antibodies. eIF4E was also purified from liver extracts with m7GTP-Sepharose and analyzed similarly by immunoblotting (left side, bottom panels). The asterisks indicate nonspecific signals (ns). (B) LPS-stimulated activation of Mnk1 and upregulation of eIF4E phosphorylation. Mnk1-Mnk2 KO mice (8 weeks old) received an intraperitoneal injection of LPS (100 μg/g of body weight, from E. coli O55:B5; Sigma). Sixty minutes after injection, the spleen and liver were dissected, and their extracts were analyzed by immunoblotting with the indicated antibodies. (C) Insulin-stimulated activation of Mnk1 and upregulation of eIF4E phosphorylation. Following an overnight fast, Mnk1-Mnk2 KO mice (8 weeks old) were injected intravenously with human insulin (100 mU/g of body weight, Novolin R; Novo Nordisk). Fifteen minutes after injection, the femoral muscle and liver were dissected, and their extracts were analyzed by immunoblotting with the indicated antibodies. Circled P, phospho.

Article Snippet: For the immunopurification of eIF4E, 1 μg of an anti-eIF4E monoclonal antibody (clone P-2; Santa Cruz) and 20 μl of protein G-Sepharose were added to 0.4 ml of the cell extract and incubated for 1 h at 4°C.

Techniques: Western Blot, Purification, Activation Assay, Injection