Journal: Cell reports

Article Title: Serum- and glucocorticoid-induced kinase drives hepatic insulin resistance by directly inhibiting AMP-activated protein kinase

doi: 10.1016/j.celrep.2021.109785

Figure Lengend Snippet: (A) Fat mass of control (n = 23) and Sgk1 Lko (n = 23) mice fed a HFD. (B–D) Liver weight (B), liver triglyceride (C), and liver cholesterol levels (D) from control (n = 12) and Sgk1 Lko (n = 13) mice fed a HFD for 16 weeks and starved for 12 h overnight. (E) Hepatic gluconeogenic gene mRNA levels in control (n = 7) and Sgk1 Lko mice (n = 5) fed with HFD for 16 weeks and starved for 12 h overnight. (F) mRNA levels of gluconeogenesis genes in control versus Sgk1 Lko primary hepatocytes (n = 5 biological replicates per group). (G) Glucose production under basal and cAMP/dexamethasone (dex) treatment conditions in control versus Sgk1 Lko primary hepatocytes (n = 9 biological replicates per group). (H and I) Hepatic lipogenesis and fatty acid oxidation gene mRNA levels in control (n = 12) and Sgk1 Lko (n = 8) male mice. Mice were fed with HFD for 16 weeks and starved for 12 h overnight prior to collecting liver tissue. (J) Decreased mTORC1 target phosphorylation in livers of Sgk1 Lko mice as evident by decreased phospho-p70S6K Thr389 and phospho-4EBP1 Ser65/Thr37/46 . See also and for information on mouse sex, age, n, and replication. *p < 0.05 and **p < 0.01 by two-way ANOVA (A, E, F, and H) or t test (B–D and G). All bars indicate mean and SEM.

Article Snippet: Sodium Palmitate is from NU-CHEK Prep Inc. For western blotting, anti-Flag M2 (RRID:AB_259529) antibody was purchased from Sigma, antibodies against SGK1 (RRID:AB_2687476), SGK2 (RRID:AB_10828732), SGK3 (RRID:AB_10949507), HSP90 (RRID:AB_2233307), Akt (RRID:AB_915783), p-Akt (Thr308) (RRID:AB_2255933), p-Akt (Ser473) (RRID:AB_2315049), NDRG1 (RRID: AB_11140640), p-NDRG1(Thr346) (RRID:AB_10693451), FoxO1 (RRID:AB_2106495), p-FoxO1/3 (Thr24/32) (RRID:AB_2106814), S6K (RRID:AB_390722), p-S6K (Thr389) (RRID:AB_2269803), S6 (RRID:AB_331355), p-S6 (S240/244) (RRID:AB_10694233), 4EBP1 (RRID:AB_2097841), p-4EBP1 (Thr37/46) (RRID:AB_560835), p-4EBP1 (Ser65) (RRID:AB_330947), AMPKα (RRID:AB_10624867), p-AMPKα (Thr172) (RRID:AB_331250), p-AMPKα(Ser485/491) (RRID:AB_331250), ACC1 (RRID:AB_2219397), p-ACC1 (Ser79) (RRID:AB_330337), Raptor (RRID:AB_561245)and p-Raptor (Ser792) (RRID:AB_2249475) were obtained from Cell Signaling Technology.

Techniques:

Journal: Cell reports

Article Title: Serum- and glucocorticoid-induced kinase drives hepatic insulin resistance by directly inhibiting AMP-activated protein kinase

doi: 10.1016/j.celrep.2021.109785

Figure Lengend Snippet: (A) AMPK inhibitor Compound C negates decreased glucose output in Sgk1 Lko primary hepatocytes (n = 9 replicates per group). (B) Compound C eliminates increased Akt phosphorylation in Sgk1 Lko primary hepatocytes, quantified in the right panel (n = 3 replicates per group). (C) Adenoviral-mediated expression of AMPKα2 Ser491Ala increases insulin-stimulated Akt Ser473/Thr308 phosphorylation in control but not in Sgk1 Lko primary hepatocytes. (D) I.p. glucose tolerance tests in control and Sgk1 Lko mice with hepatic overexpression of GFP or AMPKα1 Ser485Ala (n = 7 for control;GFP, n = 10 for control;AMPKα Ser485Ala , n = 7 for Sgk1 Lko ;GFP, n = 9 for Sgk1 Lko ;AMPKα Ser485Ala ). (E) I.p. insulin tolerance tests in control and Sgk1 Lko mice with hepatic overexpression of GFP or AMPKα1 Ser485Ala (n = 7 mice per group). (F) Insulin-stimulated phosphorylation of Akt and Foxo in control and Sgk1 Lko mice with AAV-mediated hepatic overexpression of GFP or AMPKα1 Ser485Ala . (G) Phosphorylation of mTORC1 substrates S6K and 4EBP1 in control and Sgk1 Lko mice with hepatic overexpression of GFP or AMPKα1 Ser485Ala . See also for information on mouse sex, age, n, and replication. *p < 0.05 and **p < 0.01 (NS, not significant) by two-way ANOVA (A, B, D, and E). All bars indicate mean and SEM.

Article Snippet: Sodium Palmitate is from NU-CHEK Prep Inc. For western blotting, anti-Flag M2 (RRID:AB_259529) antibody was purchased from Sigma, antibodies against SGK1 (RRID:AB_2687476), SGK2 (RRID:AB_10828732), SGK3 (RRID:AB_10949507), HSP90 (RRID:AB_2233307), Akt (RRID:AB_915783), p-Akt (Thr308) (RRID:AB_2255933), p-Akt (Ser473) (RRID:AB_2315049), NDRG1 (RRID: AB_11140640), p-NDRG1(Thr346) (RRID:AB_10693451), FoxO1 (RRID:AB_2106495), p-FoxO1/3 (Thr24/32) (RRID:AB_2106814), S6K (RRID:AB_390722), p-S6K (Thr389) (RRID:AB_2269803), S6 (RRID:AB_331355), p-S6 (S240/244) (RRID:AB_10694233), 4EBP1 (RRID:AB_2097841), p-4EBP1 (Thr37/46) (RRID:AB_560835), p-4EBP1 (Ser65) (RRID:AB_330947), AMPKα (RRID:AB_10624867), p-AMPKα (Thr172) (RRID:AB_331250), p-AMPKα(Ser485/491) (RRID:AB_331250), ACC1 (RRID:AB_2219397), p-ACC1 (Ser79) (RRID:AB_330337), Raptor (RRID:AB_561245)and p-Raptor (Ser792) (RRID:AB_2249475) were obtained from Cell Signaling Technology.

Techniques: Expressing, Over Expression

Journal: Cell reports

Article Title: Serum- and glucocorticoid-induced kinase drives hepatic insulin resistance by directly inhibiting AMP-activated protein kinase

doi: 10.1016/j.celrep.2021.109785

Figure Lengend Snippet:

Article Snippet: Sodium Palmitate is from NU-CHEK Prep Inc. For western blotting, anti-Flag M2 (RRID:AB_259529) antibody was purchased from Sigma, antibodies against SGK1 (RRID:AB_2687476), SGK2 (RRID:AB_10828732), SGK3 (RRID:AB_10949507), HSP90 (RRID:AB_2233307), Akt (RRID:AB_915783), p-Akt (Thr308) (RRID:AB_2255933), p-Akt (Ser473) (RRID:AB_2315049), NDRG1 (RRID: AB_11140640), p-NDRG1(Thr346) (RRID:AB_10693451), FoxO1 (RRID:AB_2106495), p-FoxO1/3 (Thr24/32) (RRID:AB_2106814), S6K (RRID:AB_390722), p-S6K (Thr389) (RRID:AB_2269803), S6 (RRID:AB_331355), p-S6 (S240/244) (RRID:AB_10694233), 4EBP1 (RRID:AB_2097841), p-4EBP1 (Thr37/46) (RRID:AB_560835), p-4EBP1 (Ser65) (RRID:AB_330947), AMPKα (RRID:AB_10624867), p-AMPKα (Thr172) (RRID:AB_331250), p-AMPKα(Ser485/491) (RRID:AB_331250), ACC1 (RRID:AB_2219397), p-ACC1 (Ser79) (RRID:AB_330337), Raptor (RRID:AB_561245)and p-Raptor (Ser792) (RRID:AB_2249475) were obtained from Cell Signaling Technology.

Techniques: Plasmid Preparation, Recombinant, Magnetic Beads, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Sequencing, Software

Journal: eLife

Article Title: UBE3A-mediated p18/LAMTOR1 ubiquitination and degradation regulate mTORC1 activity and synaptic plasticity

doi: 10.7554/eLife.37993

Figure Lengend Snippet: Antibodies, chemicals, and plasmids used in this study

Article Snippet: Antibody , anti-p-4EBP1 Ser65 (rabbit polyclonal) , Cell Signaling Technology , 9451; RRID: AB_330947 , (1:1000).

Techniques: Recombinant

Journal: eLife

Article Title: UBE3A-mediated p18/LAMTOR1 ubiquitination and degradation regulate mTORC1 activity and synaptic plasticity

doi: 10.7554/eLife.37993

Figure Lengend Snippet:

Article Snippet: Antibody , anti-p-4EBP1 Ser65 (rabbit polyclonal) , Cell Signaling Technology , 9451; RRID: AB_330947 , (1:1000).

Techniques: Control, shRNA, Virus, Sequencing, Recombinant, Mutagenesis, Ubiquitin Proteomics, Software

Journal: Cell reports

Article Title: Synonymous codon usage regulates translation initiation

doi: 10.1016/j.celrep.2023.113413

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-eIF4E , MBL Lifescience , Cat# RN001P, lot 004; RRID:AB_1570634.

Techniques: Imaging, Luciferase, Control, Virus, Recombinant, Transfection, Expressing, Purification, Reverse Transcription, Reporter Assay, SYBR Green Assay, Membrane, Hybridization, Protease Inhibitor, Quantitation Assay, CRISPR, Software

Journal:

Article Title: Novel Regulatory Factors Interacting with the Promoter of the Gene Encoding the mRNA Cap Binding Protein (eIF4E) and Their Function in Growth Regulation

doi:

Figure Lengend Snippet: Identification of sites within the eIF4E promoter necessary for promoter activity. (Top) General scheme of linker-scanning mutations. (Bottom) The indicated eIF4E-CAT linker-scanner constructs were transfected into HeLa cells and analyzed for CAT activity. The effect of the mutation on CAT expression compared with that of the unaltered eIF4E-CAT construct is presented as the mean and standard error. The mean and standard error are based on three transfections, each performed in duplicate (n = 6 for each determination).

Article Snippet: The membrane was cut according to the molecular weights of the proteins to be identified, and the identical blot was incubated with anti-c-Myc (9E10; Santa Cruz), anti-actin (N-350; Boehringer), or anti-eIF4E (rabbit polyclonal; gift of Nahum Sonenberg) antibodies.

Techniques: Activity Assay, Construct, Transfection, Mutagenesis, Expressing

Journal:

Article Title: Novel Regulatory Factors Interacting with the Promoter of the Gene Encoding the mRNA Cap Binding Protein (eIF4E) and Their Function in Growth Regulation

doi:

Figure Lengend Snippet: Comparison of sequences from the mouse and human eIF4E promoter. The sequences of the mouse and human promoter regions extending 5′ to the PstI site used to make peIF4E-CAT are compared. The mouse and human promoters are markedly similar in the regions of the human promoter used in these studies. MB1 and MB2 identify the two myc boxes previously identified in this promoter (35). Exon 1 is shaded. The CCAAT box and the three linker sites critical to promoter functions are boxed and indicated.

Article Snippet: The membrane was cut according to the molecular weights of the proteins to be identified, and the identical blot was incubated with anti-c-Myc (9E10; Santa Cruz), anti-actin (N-350; Boehringer), or anti-eIF4E (rabbit polyclonal; gift of Nahum Sonenberg) antibodies.

Techniques:

Journal:

Article Title: Novel Regulatory Factors Interacting with the Promoter of the Gene Encoding the mRNA Cap Binding Protein (eIF4E) and Their Function in Growth Regulation

doi:

Figure Lengend Snippet: EMSA experiments identify a unique protein binding region within the eIF4E proximal promoter sequences. (A) LS1 through LS5 digested with MscI and XhoI generated a series of insertions across the promoter. These oligonucleotides and the corresponding sequences from unaltered eIF4E-CAT were radiolabeled and analyzed by standard EMSA. The indicated cold competitor oligonucleotide (1000× Cold) contained the wild-type eIF4E oligonucleotide to evaluate specificity. The sites where linker sequences replace endogenous sequence are written in lowercase and are underlined for emphasis throughout this figure. (B) LS2 through LS7 were digested with XbaI and XhoI, generating a series of 5′ deletion mutants. These oligonucleotides were radiolabeled and EMSAs were performed as above. The indicated cold competitor oligonucleotide (1000× Cold) contained the wild-type eIF4E oligonucleotide. (C) LS1 through LS5 were digested with MscI and XbaI, generating a series of 3′ deletion mutants. These oligonucleotides and the corresponding sequences from unaltered eIF4E-CAT were radiolabeled and EMSAs were performed as above. The indicated cold competitor oligonucleotide (1000× Cold) contained wild-type eIF4E.

Article Snippet: The membrane was cut according to the molecular weights of the proteins to be identified, and the identical blot was incubated with anti-c-Myc (9E10; Santa Cruz), anti-actin (N-350; Boehringer), or anti-eIF4E (rabbit polyclonal; gift of Nahum Sonenberg) antibodies.

Techniques: Protein Binding, Generated, Sequencing

Journal:

Article Title: Novel Regulatory Factors Interacting with the Promoter of the Gene Encoding the mRNA Cap Binding Protein (eIF4E) and Their Function in Growth Regulation

doi:

Figure Lengend Snippet: Core binding activity at the eIF4E binding site differs from known initiator elements in cross-competition experiments. (A) Core binding seen at a unique binding site in the eIF4E promoter is modified by interactions at flanking sites. The LS543 oligonucleotide and the LS3 oligonucleotide were directly radiolabeled. LS4 and eIF4E-CAT were digested with XbaI and XhoI and radiolabeled. The resulting oligonucleotides were subjected to EMSA as described in the text. Cold competitor oligonucleotide (row 100×) was included to demonstrate specificity. Lines in the schematic below the gel show what portion of the whole region between −59 and +3 is included in each of the indicated oligonucleotides. (B) Core binding activity at the eIF4E binding site differs from known initiator elements. The LS5 and LS3 oligonucleotides were directly radiolabeled. LS2 and LS6 were digested with XbaI and XhoI and radiolabeled. The resulting oligonucleotides were subjected to EMSA as described above. Cold competitor oligonucleotides (row 1000×) were used to determine specificity (see Fig. ​Fig.11 for identification of competitors).

Article Snippet: The membrane was cut according to the molecular weights of the proteins to be identified, and the identical blot was incubated with anti-c-Myc (9E10; Santa Cruz), anti-actin (N-350; Boehringer), or anti-eIF4E (rabbit polyclonal; gift of Nahum Sonenberg) antibodies.

Techniques: Binding Assay, Activity Assay, Modification

Journal:

Article Title: Novel Regulatory Factors Interacting with the Promoter of the Gene Encoding the mRNA Cap Binding Protein (eIF4E) and Their Function in Growth Regulation

doi:

Figure Lengend Snippet: Decreased eIF4E expression during myeloid differentiation correlates with decreased levels of the 4E regulatory factors, c-Myc and protein synthesis. (A and B) For Southwestern analyses, protein lysates from U937 (A) and HL60 (B) cells were prepared with Laemmli buffer at 0, 3, 6, 24, 48, and 72 h after the addition of TPA. The lysates (50 μg) at the indicated time points were analyzed with the radioactively labeled LS3 trimer oligonucleotide in a Southwestern assay (SW panels). Size markers (lane SM) are indicated. For Northern blots, total cellular RNA was harvested from U937 (second through fourth panels in panel A) and HL60 (second through fourth panels in panel B) cells after the addition of TPA. RNA was size fractionated, run on formaldehyde-agarose gels, blotted, and hybridized with eIF4E, c-myc, or GAPDH plasmid fragments (4E, myc, and GAPDH, respectively). The protein lysates used for the Southwestern analyses were additionally run on 10% denaturing polyacrylamide gels, blotted, and probed with anti-eIF4E, anti-c-Myc, and anti-actin antibodies for the U937 (fifth through seventh panels [4E, myc, and actin] in panel A) and HL60 (fifth through seventh panels [4E, myc, and actin] in panel B) cells. (C through F) U937 (C and E) and HL60 (D and F) cells were pulse-labeled for 3 h with [35S]methionine and [3H]thymidine at the indicated time points. Aliquots of protein lysates at each time point were harvested directly in Laemmli buffer and run on 10% denaturing polyacrylamide gels to simultaneously evaluate protein synthesis rates of multiple individual proteins (C and D). Counts incorporated during pulse labeling were further evaluated by trichloroacetic acid precipitation of cell lysates (E and F). [35S]methionine (solid bars; y axis on right) and [3H]thymidine (open bars; y axis on left) incorporation is displayed as the mean and standard deviation of four determinations at each time point to evaluate the regulation of net protein synthesis (35S) and DNA synthesis (3H) during differentiation.

Article Snippet: The membrane was cut according to the molecular weights of the proteins to be identified, and the identical blot was incubated with anti-c-Myc (9E10; Santa Cruz), anti-actin (N-350; Boehringer), or anti-eIF4E (rabbit polyclonal; gift of Nahum Sonenberg) antibodies.

Techniques: Expressing, Labeling, Northern Blot, Plasmid Preparation, TCA Precipitation, Standard Deviation, DNA Synthesis

Journal:

Article Title: Novel Regulatory Factors Interacting with the Promoter of the Gene Encoding the mRNA Cap Binding Protein (eIF4E) and Their Function in Growth Regulation

doi:

Figure Lengend Snippet: The polypyrimidine element in eIF4E is related to other initiator regions but contains significant differences. (A) Sequence is conserved at polypyrimidine (pPy) element 3 between mouse and human sequences. This sequence is further compared with the consensus sequence for an initiator region and for the YY1 element. Underlining indicates nucleotides required for YY1 binding which differ in the eIF4E element. Shaded boxes indicate nucleotides normally required for initiator binding which differ in the eIF4E element. (B) Model for potential direct and indirect effects of c-myc on the eIF4E promoter. c-myc may activate the eIF4E promoter by directly interacting with either or both of its myc boxes (LS8 and LS23). Alternatively, c-myc may indirectly regulate eIF4E through regulation of proteins binding at the LS3 site.

Article Snippet: The membrane was cut according to the molecular weights of the proteins to be identified, and the identical blot was incubated with anti-c-Myc (9E10; Santa Cruz), anti-actin (N-350; Boehringer), or anti-eIF4E (rabbit polyclonal; gift of Nahum Sonenberg) antibodies.

Techniques: Sequencing, Binding Assay