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

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:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 1. CBP80 and eIF4E are bound to mRNA in both nuclear and cytoplasmic cell fractions. (A) Structure of the pmCMV-Gl test plasmids, which harbor either a nonsense-free Gl allele that terminates translation at codon 147 (Norm) or a Gl allele carrying a TAG nonsense codon at position 39 (Ter). (B) Cos cells were transiently transfected with a test pmCMV-Gl plasmid and the reference plasmid phCMV-MUP. Nuclear (N) and cytoplasmic (C) fractions were then immunopurified under conditions that preserved RNP using either normal rabbit serum (NRS), anti-CBP80 antibody (α-CBP80) or anti-eIF4E antibody (α-eIF4E), and subjected to western blotting using anti-CBP80 antibody or anti-eIF4E antibody. Nuclear fractions were free of cytoplasm as evidenced by the absence of detectable reactivity with anti-eIF4A antibody (data not shown). For easy visualization, bands corresponding to antibody are not shown. (C) The levels of Gl and MUP mRNAs were quantitated in RNA prepared from each immunopurification using RT–PCR. The leftmost five lanes, which analyze decreasing amounts of RNA before immunopurification (– IP), demonstrate that the conditions of RT–PCR were quantitative. Numbers below the figure represent the level of Gl mRNA normalized to the level of MUP mRNA, where normalized levels of nonsense-free (Norm) mRNA in each anti-CBP80 and anti-eIF4E immunopurification of each fraction are defined as 100%. Results are representative of two independently performed experiments where the efficiency of NMD varied by no more than 12%. The immunopurification using anti-eIF4E and cytoplasmic sample was less efficient for the transfection using pmCMV-Gl Norm than for the transfection using pmCMV-Gl Ter, explaining the reduced levels of eIF4E detected by western blotting (B) and RNA detected by RT–PCR (C).

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Transfection, Plasmid Preparation, Western Blot, Immu-Puri, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 2. CBP80 but not eIF4E is detectably bound to intron-containing pre-mRNA. (A) As in Figure 1, except that only nuclear fractions and Gl pre-mRNA rather than Gl mRNA were analyzed. Furthermore, mouse anti-eIF4E antibody was used instead of rabbit anti-eIF4E antibody and mouse IgG served to control for non-specific immunopurification using mouse anti-eIF4E antibody. (B) As in (A) except that rabbit anti-eIF4E antibody was used in place of mouse anti-eIF4E antibody and the analysis of MUP mRNA was omitted. (C) As in (A) except that SV40 pre-mRNA and mRNA were analyzed. Results are representative of other independently performed experiments, including those using rabbit anti-eIF4E antibody and NRS as a control. Taking the sum of Gl or SV40 pre-mRNA immunopurified by anti-CBP80 antibody and anti-eIF4E antibody in each panel as 100%, the amount of CBP80-bound pre-mRNA was 96 ± 6% and the amount of eIF4E-bound pre-mRNA was 4 ± 6%.

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Immu-Puri

Journal:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 3. CBP80 but neither eIF4E nor Upf2 immunopurifies with the CTD. The nuclear fraction of 32 × 106 untransfected Cos cells was immunopurified using either NRS or antibody to the C-terminal domain of RNA polymerase II (α-CTD), and protein from either 8 × 105 cells (CTD) or 32 × 105 cells (CBP80, eIF4E and Upf2) was subjected to western blotting using anti-CTD, anti-CBP80, anti-eIF4E or anti-Upf2 antibody. The three leftmost lanes, which analyze 2-fold dilutions of nuclear protein from 1.6 × 105 cells (CTD and CBP80) or 6.4 × 105 cells (eIF4E and Upf2) before immunopurification (– IP), demonstrate that the conditions of western blotting were semi-quantitative. The two forms of CTD probably differ in the degree of phosphorylation (Dubois et al., 1994). Results are representative of 2–4 independently performed experiments, depending on the antibody used in western blotting.

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Western Blot, Immu-Puri

Journal:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 5. RNPS1, Y14, SRm160, REF/Aly and Upf2 co-immunopurify with Upf3X in an RNase-insensitive manner, while RNPS1, Y14, REF/Aly, TAP, Upf3X and Upf2 co-immunopurify with CBP80 in an RNase-sensitive manner. As in Figure 4 except that an additional immunopurification using anti-Upf3/3X was performed, total (T) lysate was immunopurified using anti-CBP80 antibody, RNase was added to half of each sample prior to immunopurification, RNA as well as protein were prepared, and western blot analyses of eIF4E and DEK were omitted. (A) RT–PCR quantitation of the level of β-actin mRNA before or after immunopurification using anti-Upf3/3X antibody in order to demonstrate the efficiency of RNase treatment. Prior to immununopurification, half of the sample was treated with RNase (+), and the other half was not treated with RNase (–). (B) Western blot analysis of protein immunopurified using anti-Upf3/3X antibody from nuclear and cytoplasmic fractions. (C) Western blot analysis of protein immunopurified using anti-CBP80 antibody from total cell extract.

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Immu-Puri, Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitation Assay

Journal:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 4. Nuclear and cytoplasmic CBP80, unlike nuclear and cytoplasmic eIF4E, co-immunopurify with RNPS1, Y14, SRm160, REF/Aly, TAP, Upf3X and Upf2. Nuclear and cytoplasmic fractions of 32 × 106 untransfected Cos cells were immunopurified using anti-CBP80 antibody, anti-eIF4E antibody or, as a control, NRS. Immunopurified protein from 8 × 105 or 32 × 105 cells was then analyzed by western blotting using the antibodies specified. The three leftmost lanes, which analyze 2-fold dilutions of nuclear protein from 8 × 105 or 32 × 105 cells before immunopurification (– IP), demonstrate that the conditions of western blotting were semi-quantitative. Notably, Upf3 probably co-migrates with the heavy chain of anti-hUpf3/3X antibody and, therefore, was not assayable.

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Western Blot, Immu-Puri

Journal:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 6. Model for the dynamics of mRNP structure in mammalian cells as a consequence of pre-mRNA capping, pre-mRNA splicing and mRNA export. In the nucleus, CBP80-bound pre-mRNA is spliced to generate CBP80-bound mRNA. This mRNA is bound by an EJC (dark gray shapes), consisting of RNPS1, Y14, SRm160 and REF/Aly, located 20–24 nucleotides upstream of the exon–exon junction. The EJC recruits the NMD factor Upf3/3X and the mRNA export factor TAP. Some mRNPs (possibly those subject to nucleus-associated NMD) undergo remodeling while associated with the nucleus (nucleus-associated remodeling). Other mRNPs (including those subject to cytoplasmic NMD) undergo remodeling after export to the cytoplasm (cytoplasmic remodeling). In both types of remodeling, CBP80 is replaced by eIF4E and the EJC is lost. Data indicate that the mostly perinuclear Upf2 protein is recruited by the EJC at a point when mRNA co-purifies with nuclei. In theory, recruitment could occur during mRNA export. However, if nucleus-associated remodeling takes place in the nucleoplasm, it is conceivable that an as yet undetected pool of Upf2 is nucleoplasmic. The single exon–exon junction exemplified is likely to typify all splicing-generated exon–exon junctions, regardless of position relative to the mRNA 3′ end.

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Generated

Journal: The American Journal of Pathology

Article Title: Tumor Necrosis Factor Receptor-2 Signals Clear-Cell Renal Carcinoma Proliferation via Phosphorylated 4E Binding Protein-1 and Mitochondrial Gene Translation

doi: 10.1016/j.ajpath.2024.02.019

Figure Lengend Snippet: pSer65 -4EBP1 is abundantly expressed in ccRCC tumor and co-localizes with tumor necrosis factor receptor (TNFR) 2. Representative confocal images of single-immunofluorescence staining of ccRCC tumor grades (G1 to G4) and corresponding nontumor tissue (NK). A: Top panels: Staining for total 4EBP1 (t4EBP1). A marked signal was detected in NK, confined to normal tubular cells (t), some isolated cells within glomeruli (Glom), and the interstitium ( red arrowheads ). A similar pattern and intensity were found in tumor cells in all ccRCC grades. Bottom panels: Staining for pSer65 -4EBP1. Signal was undetectable in NK and infrequently present in G1 ccRCC tumor cells but accumulated with increasing intensity in ccRCC according to malignant grade (G2 to G4). Signal was mainly cytoplasmic but also found infrequently in the nucleus of some tumor cells. Inset: low-power image; arrowhead indicates the panel labeled super-res shows a high-power image of 4E-BP1 staining in the nuclei taken by super-resolution microscopy. B: Signal intensity per cell was quantified as corrected total cell fluorescence (CTCF). Two-way analysis of variance was performed. C: Immunoblot of lysates from three independent ccRCC G4 tumor resections (T) alongside corresponding nontumor kidney (N) from the same tissue, showing an increased abundance of pSer65 -4EBP1 in all three tumors compared with normal tissue. β-Actin and Ponceau S stain show protein loading. Bar graph shows the relative intensity of pSer65 -4EBP1 band normalized to N for each pair. D: Representative confocal images of ccRCC G4 staining showing co-localization of signal for pSer65 -4EBP1 and TNFR2 in tumor cells and in infiltrating cells within the interstitium ( inset ; arrowheads ). Data are expressed as means ± SD ( B ). n = 3 per group with similar results ( A , B , and D ). ∗∗∗ P < 0.001 versus NK and G1; ∗∗∗∗ P < 0.0001 versus NK, G1, and G2. Scale bars: 50 μm ( A and D , main images ); 25 μm ( D , inset ). Original magnification: ×40 ( A ); ×63 ( A , super-res image , and D , main images and inset ). NS, not significant.

Article Snippet: Rabbit anti–phosphorylated 4EBP1 S65 , AP0032 , ABclonal (Woburn, MA) , IF.

Techniques: Immunofluorescence, Staining, Isolation, Labeling, Super-Resolution Microscopy, Fluorescence, Western Blot

Journal: The American Journal of Pathology

Article Title: Tumor Necrosis Factor Receptor-2 Signals Clear-Cell Renal Carcinoma Proliferation via Phosphorylated 4E Binding Protein-1 and Mitochondrial Gene Translation

doi: 10.1016/j.ajpath.2024.02.019

Figure Lengend Snippet: Activation of tumor necrosis factor receptor (TNFR) 2 by TNFR2-specific mutein (R2TNF) induces expression of pSer65 -4EBP1 and its co-localization with TNFR2 in tumor cells in organ cultures of ccRCC. A: Representative confocal images of combined immunofluorescence in low-grade ccRCC organ culture sections stained for total (t) 4EBP1. A similar signal intensity was found in tumor cells (identified using the tumor marker RCC-MA) in control [untreated (UT)] and R2TNF-treated cultures. B: In comparison, R2TNF induced expression of pSer65 -4EBP1 in tumor cells with a weak infrequent signal in controls. A signal for pSer65 -4EBP1 is also seen in endothelial cells in blood vessels (Bv; white arrows ), negative for RCC-MA. Numbers indicate percentage co-localization in tumor cells. C: pSer65 -4EBP1 and TNFR2 co-localization in tumor cells, in vascular endothelial cells (Bv; white arrow s ), and in infiltrating cells ( yellow arrowheads ). Inset : Cosignal in a bilobed tumor cell in another area on the same section. Data are given as means ± SD ( A and B ). n = 3 per group with similar results ( A and B ). Scale bars: 50 μm ( A – C, main image s ); 10 μm ( C , inset ). Original magnification: ×40 ( A – C , main images ); ×63 ( C , inset ).

Article Snippet: Rabbit anti–phosphorylated 4EBP1 S65 , AP0032 , ABclonal (Woburn, MA) , IF.

Techniques: Activation Assay, Expressing, Immunofluorescence, Organ Culture, Staining, Marker, Control, Comparison

Journal: The American Journal of Pathology

Article Title: Tumor Necrosis Factor Receptor-2 Signals Clear-Cell Renal Carcinoma Proliferation via Phosphorylated 4E Binding Protein-1 and Mitochondrial Gene Translation

doi: 10.1016/j.ajpath.2024.02.019

Figure Lengend Snippet: Tumor necrosis factor receptor (TNFR) 2–specific mutein (R2TNF) induces co-localization of pSer65 -4EBP1 and TNFR2 in mitochondria (m). Representative panels of high-power confocal images of combined immunofluorescence (IF). A and B: pSer65 -4EBP1 and heat shock protein (HSP) 60 (mitochondrial matrix protein; A ) or TNFR2 and HSP60 ( B ) in low-grade ccRCC organ culture sections. Images show R2TNF-induced co-localization of the two proteins in mitochondria in tumor cells ( arrowheads ). C and D: Super-resolution images ( super-res) of tumor cells show mitochondrial co-localization of the two proteins ( white arrowheads ; C ), confirmed by triple IF ( D ), which shows pSer65 -4EBP1/TNFR2/HSP60 + ( arrows ) within a tumor cell ( white dotted lines ). E: Representative immunogold electron micrographs show presence of pSer65 -4EBP1 and TNFR2 (15-nm colloidal gold) in mitochondria, labeled with HSP60 (5-nm colloidal gold) on R2TNF-treated low-grade ccRCC organ sections ( white arrows ). F: The close proximity of pSer65 -4EBP1 ( red arrows ) and TNFR2 ( blue arrows ) in mitochondria and the presence of TNFR2 on the cell surface compared with untreated (UT) cultures, which show a rare signal. n = 3 per group with similar results ( A – F ). Scale bars: 25 μm ( A and B ); 75 μm ( C and D ); 200 nm ( E and F, left and right panels ); 500 nm ( F , middle panel ). Original magnifications: ×40 ( A and B ); ×63 ( C and D ). N, nucleus.

Article Snippet: Rabbit anti–phosphorylated 4EBP1 S65 , AP0032 , ABclonal (Woburn, MA) , IF.

Techniques: Immunofluorescence, Organ Culture, Labeling

Journal: The American Journal of Pathology

Article Title: Tumor Necrosis Factor Receptor-2 Signals Clear-Cell Renal Carcinoma Proliferation via Phosphorylated 4E Binding Protein-1 and Mitochondrial Gene Translation

doi: 10.1016/j.ajpath.2024.02.019

Figure Lengend Snippet: Tumor necrosis factor receptor (TNFR) 2–specific mutein (R2TNF) induces co-localization of pSer65 -4EBP1 and TNFR2 with cytochrome c oxidase (Cox) subunits. A: Representative confocal images of combined immunofluorescence of pSer65 -4EBP1 or TNFR2 with Cox1, Cox4, or Cox5b in low-grade ccRCC organ culture sections show R2TNF-induced cosignal of Ser65 -4EBP1 or TNFR2 and Cox1, Cox4, and Cox5. B: High-power images show R2TNF-induced cosignal of Ser65 -4EBP1 or TNFR2 and Cox1. C: Quantification of signal intensity of R2TNF-induced cosignal compared with untreated (UT) presented as corrected total cell fluorescence (CTCF). Two-way analysis of variance was performed. Data are given as means ± SD ( C ). n = 3 per group with similar results ( C ). ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001 versus controls. Scale bars: 50 μm ( A ); 25 μm ( B , top panels ); 10 μm ( B , bottom panels ). Original magnification: ×40 ( A ); ×63 ( B ).

Article Snippet: Rabbit anti–phosphorylated 4EBP1 S65 , AP0032 , ABclonal (Woburn, MA) , IF.

Techniques: Immunofluorescence, Organ Culture, Fluorescence

Journal: The American Journal of Pathology

Article Title: Tumor Necrosis Factor Receptor-2 Signals Clear-Cell Renal Carcinoma Proliferation via Phosphorylated 4E Binding Protein-1 and Mitochondrial Gene Translation

doi: 10.1016/j.ajpath.2024.02.019

Figure Lengend Snippet: Tumor necrosis factor receptor 2–specific mutein (R2TNF)–induced cell proliferation and pSer65 -4EBP1 expression in tumor cells are reduced by mammalian target of rapamycin (mTOR) inhibitors. A: Representative confocal images of combined immunofluorescence for pSer65 -4EBP1 (green) and proliferative marker phosphorylated-histone S10 (pH3 S10 ; red) in low-grade ccRCC organ culture either left untreated (UT) or treated with R2TNF for 3 hours at 37°C show a significant increase in proliferative tumor cells (pH3 S10+ ) also positive for pSer65 -4EBP1 in R2TNF-treated cultures compared with UT. B: Similar cultures pretreated with mTOR inhibitors Torin 2, Ku63794, or rapamycin for 1 hour before R2TNF addition show a significant reduction in pSer65 -4EBP1 + /pH3 S10+ tumor cells, more pronounced in cultures pretreated with Torin 2 and Ku63794 than rapamycin. C: Quantification of tumor cell proliferation as a proliferative index (percentage positive of total cells) in UT or R2TNF-treated organ cultures of ccRCC (RCCoC) versus staining in normal tubular cells in organ cultures of nontumor tissue (NKoC) and in the absence or presence of the mTOR inhibitors. D: Quantification of double-stained pSer65 -4EBP1 + /pH3 S10 positive tumor cells in UT and R2TNF-treated cultures ± mTOR inhibitors. Two-way analysis of variance was performed. Data are given as means ± SD ( C and D ). n = 3 per group with similar results ( C and D ). ∗ P < 0.05, ∗∗∗ P < 0.001 versus R2TNF; ∗∗∗∗ P < 0.0001 versus UT. Scale bars: 50 μm ( A ); 75 μm ( B ). Original magnifications: ×2.3 ( B ); ×40 ( C ). NS, not significant.

Article Snippet: Rabbit anti–phosphorylated 4EBP1 S65 , AP0032 , ABclonal (Woburn, MA) , IF.

Techniques: Expressing, Immunofluorescence, Marker, Organ Culture, Staining

Journal: The American Journal of Pathology

Article Title: Tumor Necrosis Factor Receptor-2 Signals Clear-Cell Renal Carcinoma Proliferation via Phosphorylated 4E Binding Protein-1 and Mitochondrial Gene Translation

doi: 10.1016/j.ajpath.2024.02.019

Figure Lengend Snippet: A model of tumor necrosis factor receptor (TNFR) 2–driven cell cycle entry in tumor cells in ccRCC. Selective ligation of TNFR2 by TNFR2-specific mutein (R2TNF) induces an increased expression of TNFR2 and phosphorylation of 4EBP1 at serine 65 ( pSer65 -4EBP1) and facilitates co-localization of the two proteins in cytoplasm and mitochondria alongside increased expression of nuclear- and mitochondrial-encoded cytochrome c oxidase (COX) subunits. pSer65 -4EBP1 is also detected in the nucleus in some tumor cells. Mammalian target of rapamycin (mTOR) inhibitors cause a significant reduction in R2TNF-mediated cell cycle activation and pSer65 -4EBP1 expression, accompanied by an increased cell death with the relative potency of Torin 2 > Ku63794 > rapamycin. mTORC, mTOR complex.

Article Snippet: Rabbit anti–phosphorylated 4EBP1 S65 , AP0032 , ABclonal (Woburn, MA) , IF.

Techniques: Ligation, Expressing, Phospho-proteomics, Activation Assay