Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Acute Regulation of Translation Initiation by Gonadotropin-Releasing Hormone in the Gonadotrope Cell Line LβT2

doi: 10.1210/me.2003-0478

Figure Lengend Snippet: LβT2 cells were serum starved overnight (A–C) and amino acid starved 1 h(B and C) before GnRH treatment at indicated doses and time points. Extracts were subjected to SDS-PAGE followed by Western blotting with the following antibodies. A, Anti-4E-BP1 reveals three electrophoretic forms (γ, β α) with the histogram representing the proportion of inactive γ-isoform relative to total 4E-BP1. B, Antiphospho-eIF4E (Ser 209) and eIF4E; histogram represents ratio of phospho-eIF4E relative to total eIF4E. C, Anti-phospho-eIF4G (Ser 1108); histogram represents values expressed as a percentage of maximal induction. Blots are representative images of each experiment. Histograms represent quantitative chemiluminescent image analysis of at least three independent experiments. The asterisks show significant difference from the control mean (P ≤ 0.05) as determined by ANOVA and post hoc Dunnett’s comparison to control test.

Article Snippet: 4E-BP1, phospho-eIF4E, and AKT antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) whereas phospho-eIF4G, -AKT, -Mnk, and -eIF4E antibodies were from Cell Signaling Technology (Beverly, MA).

Techniques: SDS Page, Western Blot, Control, Comparison

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Acute Regulation of Translation Initiation by Gonadotropin-Releasing Hormone in the Gonadotrope Cell Line LβT2

doi: 10.1210/me.2003-0478

Figure Lengend Snippet: LβT2 cells were serum starved overnight and amino acid starved for 1 h followed with 10 nm GnRH for the times shown. Extracts underwent SDS-PAGE and immunoblotted with the indicated antiserum. A, Three electrophoretic forms (γ, β α) of 4E-BP1; the histogram depicts the proportion of inactive γ-isoform relative to total 4E-BP1. B, Histogram represents proportion of phospho-eIF4E relative to total eIF4E. C, Percent of maximal eIF4G phosphorylation. Blots are representative images. Histograms are the result of quantitative chemiluminescent imaging analysis of at least three separate experiments. The asterisks show significant difference from the control mean (P ≤ 0.05) as determined by ANOVA and post hoc Dunnett’s comparison to control test.

Article Snippet: 4E-BP1, phospho-eIF4E, and AKT antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) whereas phospho-eIF4G, -AKT, -Mnk, and -eIF4E antibodies were from Cell Signaling Technology (Beverly, MA).

Techniques: SDS Page, Phospho-proteomics, Imaging, Control, Comparison

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Acute Regulation of Translation Initiation by Gonadotropin-Releasing Hormone in the Gonadotrope Cell Line LβT2

doi: 10.1210/me.2003-0478

Figure Lengend Snippet: LβT2 cells were serum starved overnight, followed by amino acid starvation for 1 h and subsequently pretreated with 10 µm PD98059, 1.0 µm LY294002, or 10 nm rapamycin for 30 min, after which cells were treated with 10 nm GnRH for 15 min. Extracts were separated by SDS-PAGE and immunoblotted with the indicated antiserum. A, Histogram represents activation of phospho-Mnk. B, Histogram represents ratio of phospho-eIF4E to total eIF4E normalized to control. Blots are representative images and histograms are the result of quantitative chemiluminescent imaging analysis of at least three separate experiments. The asterisks show significant difference from the control mean (P ≤ 0.05), as determined by ANOVA and post hoc Dunnett’s comparison to control test.

Article Snippet: 4E-BP1, phospho-eIF4E, and AKT antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) whereas phospho-eIF4G, -AKT, -Mnk, and -eIF4E antibodies were from Cell Signaling Technology (Beverly, MA).

Techniques: SDS Page, Activation Assay, Control, Imaging, Comparison

Journal: Nature communications

Article Title: Integrating fragment-based screening with targeted protein degradation and genetic rescue to explore eIF4E function.

doi: 10.1038/s41467-024-54356-1

Figure Lengend Snippet: Fig. 4 | Compound 4 inhibits eIF4G:eIF4E binding and cap-dependent transla- tion in cell lysate assays. a Lysates from SW620 cells were incubated with 1–100 µM compound 4 or 5 or positive control peptide (RIIY) for 30 min. Endo- genous eIF4E was immunoprecipitated and immunoblotted for eIF4G, 4E-BP1 and eIF4E. Quantitation of 4E-BP1 (b) or eIF4G (c) with endogenous eIF4E in SW620 and HeLa cell lysates, determined by the electro-chemiluminescent binding assay fol- lowing incubation for 30 min with DMSO vehicle (Cont), 100 µM compound 4 or 100 µM RIIY peptide. Complexes were immobilised by an eIF4E antibody and captured eIF4E, eIF4G and 4E-BP1 detected by their respective secondary anti- bodies. Values represent ratios of 4E-BP1:eIF4E or eIF4G:eIF4E electro- chemiluminescence relative to DMSO control (n = 2 biological replicates). d Electro-chemiluminescent assay for binding of eIF4G or 4E-BP1 with eIF4E in SW620 (n = 2 biological replicates), or (e) in HeLa lysates (n = 3 biological replicates, mean ± SD) following incubation for 30 min with compound 4 or 5. Results are expressed as luminescence signals relative to DMSO control. f Quantification of eIF4E:eIF4G interaction in H1299 cells by electro-chemiluminescent assay. Cell lysates treated with RIIY 4E-BP1 derived peptide or RIIG negative control peptide at 0.1–100 µM for 30 min (n = 2 biological replicates). g Quantification of the endo- genous eIF4E:eIF4G interaction in H1299 cell lysates at 0.1–100 µM (for 6 h) of compound 4 or 5, as measured by electro-chemiluminescent assay (mean ± SD from n = 3 biological replicates). h HeLa cell lysates for in vitro translation were incubated for 30 min with 1, 10, 100 µM of compound 4 or 5. Results are expressed as firefly or renilla luminescence signal normalized to DMSO control and expressed as % (mean ± SD from n = 3 biological replicates). Significance was determined using two-sided unpaired t-test comparing compound 4 to compound 5 at each concentration. Statistically significant p-values (p < 0.05) are shown on the plot and source data is located in the Source Data file.

Article Snippet: Blots were probed at 4 °C overnight for eIF4E (Cell Signaling, Cat#2067, RRID:AB_2097675), MCL1 (Cell Signaling, cat#39224, RRID:AB_2799149), monoclonal ANTI-FLAG® M2 (Sigma-Aldrich, cat#F1804, RRID:AB_262044), and vinculin (Cell Signaling, cat#13901, RRID:AB_2728768).

Techniques: Binding Assay, Incubation, Positive Control, Immunoprecipitation, Quantitation Assay, Control, Derivative Assay, Negative Control, In Vitro, Concentration Assay

Journal: Scientific Reports

Article Title: Alpha-ketoglutarate promotes skeletal muscle hypertrophy and protein synthesis through Akt/mTOR signaling pathways

doi: 10.1038/srep26802

Figure Lengend Snippet: C2C12 cells were cultured for 6 d in a differentiation medium. C2C12 myotubes were then exposed to different concentrations of AKG (0, 0.5, and 2 mM) for 48 h. ( a ) Total protein levels. ( b ) Puromycin levels detected in C2C12 myotubes by Western blot. ( c ) The phosphorylation levels of mTOR, S6, 4E-BP1, eIF4E, eIF2a, and MyHC by Western blot. ( d ) IHC analysis for MHCII and phospho-S6 in C2C12 myotubes. Data are presented as mean±S.E.M. Different superscripts “a”/“b”/“c” represent significant differences between groups ( P < 0.05), and * means P < 0.05 compared with the control. β-actin served as a housekeeping gene.

Article Snippet: The PVDF membranes were then incubated with the indicated antibodies, including rabbit anti-β-actin (Bioss) and mouse puromycin antibody 12D10 (Millipore); or rabbit anti-phospho-mTOR (Ser2481) and mTOR, rabbit anti-phosphor-P70S6K (Thr389) and P70S6K1, rabbit anti-phospho-S6 (Ser235/236) and S6, rabbit anti-phospho-4E-BP1 (Thr37/46) and 4E-BP1, rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308;), rabbit anti-eIF4E, rabbit anti-phospho-eIF4E (Ser209), rabbit anti-eIF2α, rabbit anti-phospho-eIF2α (Ser251), rabbit anti-FoxO1, and rabbit anti-phospho-FoxO1 (Ser256) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Cell Culture, Western Blot, Phospho-proteomics, Control

Journal: Scientific Reports

Article Title: Alpha-ketoglutarate promotes skeletal muscle hypertrophy and protein synthesis through Akt/mTOR signaling pathways

doi: 10.1038/srep26802

Figure Lengend Snippet: ( a ) C2C12 myotubes were treated with 2 mM AKG for 1, 2, and 4 h. The expression levels of phosphorylation of mTOR, S6K, S6, 4E-BP1, eIF2a, and eIF4E were detected by Western blot. ( b ) C2C12 cells were cultured for 6 d in a differentiation medium. mTOR inhibitor rapamycin (1 μM) was used alone or co-treated with AKG (2 mM) for 48 h. Total protein levels. ( c ) The expression of puromycin determined by Western blot after C2C12 cells were co-treated with AKG and rapamycin. ( d ) The expression levels of phosphorylation of mTOR, S6, and 4E-BP1 detected by Western blot after C2C12 cells were co-treated with AKG and rapamycin. Data are presented as mean ± SEM. *means P < 0.05 compared with the control. β-actin served as a housekeeping gene control.

Article Snippet: The PVDF membranes were then incubated with the indicated antibodies, including rabbit anti-β-actin (Bioss) and mouse puromycin antibody 12D10 (Millipore); or rabbit anti-phospho-mTOR (Ser2481) and mTOR, rabbit anti-phosphor-P70S6K (Thr389) and P70S6K1, rabbit anti-phospho-S6 (Ser235/236) and S6, rabbit anti-phospho-4E-BP1 (Thr37/46) and 4E-BP1, rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308;), rabbit anti-eIF4E, rabbit anti-phospho-eIF4E (Ser209), rabbit anti-eIF2α, rabbit anti-phospho-eIF2α (Ser251), rabbit anti-FoxO1, and rabbit anti-phospho-FoxO1 (Ser256) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, Phospho-proteomics, Western Blot, Cell Culture, Control

Journal: Scientific Reports

Article Title: Alpha-ketoglutarate promotes skeletal muscle hypertrophy and protein synthesis through Akt/mTOR signaling pathways

doi: 10.1038/srep26802

Figure Lengend Snippet: AKG (0.6 g/kg) and puromycin were co-injected for 3 h. Protein turnover associate protein expression was detected by Western blot. ( a ) The expression of puramycin was analyzed by Western blot. ( b ) The expression of MyHC and the phosphorylation levels of mTOR, S6, 4E-BP1, eIF4E, eIF2a in the gastrocnemius of mice. ( c ) The expression of Akt, MAFbx, and MuRF1. ( d ) The mRNA expression levels of protein turnover related genes were measured by qPCR. ( e ) The phosphorylation levels of Akt and mTOR 1 h after AKG injection detected by Western blot. Data are presented as mean ± SEM. *means P < 0.05 compared with the control. β-actin served as a housekeeping gene control.

Article Snippet: The PVDF membranes were then incubated with the indicated antibodies, including rabbit anti-β-actin (Bioss) and mouse puromycin antibody 12D10 (Millipore); or rabbit anti-phospho-mTOR (Ser2481) and mTOR, rabbit anti-phosphor-P70S6K (Thr389) and P70S6K1, rabbit anti-phospho-S6 (Ser235/236) and S6, rabbit anti-phospho-4E-BP1 (Thr37/46) and 4E-BP1, rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308;), rabbit anti-eIF4E, rabbit anti-phospho-eIF4E (Ser209), rabbit anti-eIF2α, rabbit anti-phospho-eIF2α (Ser251), rabbit anti-FoxO1, and rabbit anti-phospho-FoxO1 (Ser256) (Cell Signaling Technology, Beverly, MA, USA).

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