Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Proteins regulating cap-dependent translation are downregulated during total knee arthroplasty

doi: 10.1152/ajpregu.00601.2011

Figure Lengend Snippet: Increased gene expression of eIF4G and the activating transcription factor 4 (ATF4) transcription target growth arrest and DNA damage 45A (GADD45A). Transcripts for eukaryotic initiation factor 4 gamma 1 (eIF4G1) increased 19% (P = 0.048) (A), eIF4G2 increased 19% (P = 0.04) (B), and eIF4G3 increase 28% (P = 0.01) (C) from baseline to reperfusion. The transcription target of ATF4, GADD45A mRNA, significantly increased during ischemia 37% (P = 0.02) and showed a trend to increase, by 30% (P = 0.11) above baseline, during reperfusion (D). Results were normalized to GAPDH. Data are expressed as mean fold change ± SE (n = 12). *P ≤ 0.05 vs. baseline.

Article Snippet: The primary antibodies p-Akt Ser 473 (no. 9271), Akt (no. 9272), p-4E-BP1 Thr 37/46 (no. 9459), 4E-BP1 (no. 9452), p-eIF4G Ser 1108 (no. 2441), eIF4G (no. 2498), p-eIF4E Ser 209 (no. 9741), eIF4E (no. 9742), p-eIF2α Ser 51 (no. 3597), p-eEF2 Thr 56 (no. 9459), eEF2 (no. 9452), p-Mnk1 (no. 2111), and Mnk1 (no. 2195) were purchased from Cell Signaling (Beverly, MA).

Techniques: Gene Expression

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Proteins regulating cap-dependent translation are downregulated during total knee arthroplasty

doi: 10.1152/ajpregu.00601.2011

Figure Lengend Snippet: Ischemia-reperfusion inhibits multiple proteins regulating cap-dependent translation. The schematic diagram of proteins regulating signaling pathways controlling cap-dependent mRNA translation initiation and elongation. Ischemia-reperfusion inhibits cap-dependent translation initiation via inhibition of Akt-mTOR pathway and availability of 4E-BP1 to bind to and inhibit eIF4E association with eIF4G to form an active mRNA cap-binding complex (eIF4F). Ischemia-reperfusion also activates endoplasmic reticulum (ER) stress via eIF2α, which inhibits cap-dependent translation initiation. ATF4 and GADD34, each downstream components of eIF2α, are proteins involved in recovery from cell stress and are upregulated during ischemia and provide inhibitory feedback onto eIF2α. Alterations due to ischemia/reperfusion (I/R) additionally stimulate mRNA for all three isoforms of eIF4G. Together, these data may potentially provide some insight into the dramatic quadriceps atrophy (−12%) occurring within 2 wk post-TKA.

Article Snippet: The primary antibodies p-Akt Ser 473 (no. 9271), Akt (no. 9272), p-4E-BP1 Thr 37/46 (no. 9459), 4E-BP1 (no. 9452), p-eIF4G Ser 1108 (no. 2441), eIF4G (no. 2498), p-eIF4E Ser 209 (no. 9741), eIF4E (no. 9742), p-eIF2α Ser 51 (no. 3597), p-eEF2 Thr 56 (no. 9459), eEF2 (no. 9452), p-Mnk1 (no. 2111), and Mnk1 (no. 2195) were purchased from Cell Signaling (Beverly, MA).

Techniques: Protein-Protein interactions, Inhibition, Binding Assay

Journal: Biochemical and Biophysical Research Communications

Article Title: Hydrogen peroxide induces stress granule formation independent of eIF2? phosphorylation

doi: 10.1016/j.bbrc.2012.06.033

Figure Lengend Snippet: H2O2 induces the assembly of non-canonical SGs. Immunofluorescence microscopy showing non-treated U2OS cells (A; No treat), cells treated with 250 μM sodium arsenite (B; SA) or 2 mM H2O2 (C; H2O2), or cells treated with H2O2 followed emetine treatment (D; H2O2+Em). Yellow arrows indicate SGs stained with SG markers [TIA-1 (green), PABP (red) and eIF4E (blue)]. Insets show enlarged views of individual and merged channels. (E) Percentage of cells with SA- or H2O2-induced SGs that contain the indicated protein markers. The average percentage of cells with SGs is shown (n=3). Error bars indicate the standard deviation. *=P values, that were calculated by comparing the percentage of cells with SGs in SA- and H2O2-treated cells (PABP, p=0.04; eIF4E, p=0.0006; eIF4G, p=0.002; and eIF3b, p=0.004). No other protein markers (G3BP, FXR1, TIA-1, and HuR) showed statistically significant differences. (F) Percentage of cells with SA- and H2O2-induced SGs before and after treatment with emetine. Error bars indicate the standard deviations of the mean (n=3). *=P value<0.005 when comparing the percentages of SGs in SA- or H2O2-treated cells with those treated with emetine.

Article Snippet: Antibodies Mouse monoclonal antibodies to G3BP, HuR, p70 S6 kinase (SK1-hedls), eIF4E, 4E-BP1, 4E-T and PABP, rabbit polyclonal antibody to eIF4G, goat polyclonal antibodies to eIF3b, TIAR, TIA-1, or FXR1 were purchased from Santa Cruz Biotechnology.

Techniques: Immunofluorescence, Microscopy, Staining, Standard Deviation

Journal: Biochemical and Biophysical Research Communications

Article Title: Hydrogen peroxide induces stress granule formation independent of eIF2? phosphorylation

doi: 10.1016/j.bbrc.2012.06.033

Figure Lengend Snippet: H2O2 induces SGs by targeting eIF4F. (A) H2O2 causes eIF4F complex disruption and enhances eIF4E:4E-BP1 interactions. U2OS cells without (No treat) or with drug treatment (sodium aresenite (SA) and H2O2) were assembled on m7GTP-Sepharose as described in [18]. Loading control (Input) and the m7GTP-bound proteins (m7GTP-beads) were analyzed by Western Blotting using antibodies against eIF4G, eIF4A, eIF4E and 4E-BP1. (B) Western blot analysis showing the knockdown efficiency of eIF4E in U2OS cells transfected with control siRNA (si-Ctrl) or eIF4E-specific siRNA (si-4E). TIA-1 was used as a loading control. (C) Immunofluorescence microscopy showing SG assembly in U2OS cells after eIF4E depletion. Cell treated with the indicated siRNAs were left untreated (No treat, upper panels) or treated with 250 μM SA (middle panels), or 2 mM H2O2 (lower panels) and then stained with SG markers G3BP (red) and TIA-1 (green). Nuclei are stained with Hoechst. Insets show enlarged views of individual and merged channels. (D) Percentage of cells with SGs after eIF4E depletion. Error bars indicate the standard deviations of the mean (n=3, *p= 0.00036)

Article Snippet: Antibodies Mouse monoclonal antibodies to G3BP, HuR, p70 S6 kinase (SK1-hedls), eIF4E, 4E-BP1, 4E-T and PABP, rabbit polyclonal antibody to eIF4G, goat polyclonal antibodies to eIF3b, TIAR, TIA-1, or FXR1 were purchased from Santa Cruz Biotechnology.

Techniques: Disruption, Control, Western Blot, Knockdown, Transfection, Immunofluorescence, Microscopy, Staining