p70 s6k constructs  (Addgene inc)

Journal: Science signaling

Article Title: A DGKζ-FoxO-ubiquitin proteolytic axis controls fiber size during skeletal muscle remodeling

doi: 10.1126/scisignal.aao6847

Figure Lengend Snippet: WT and DGKζ KO mice were subjected to mechanical overload or sham surgery (A to D, F, and G), or a bout of maximal-intensity contractions (MIC+) or the control condition (MIC−) (E). The plantaris and tibialis anterior muscles (n) were collected at 2 days after surgery or immediately after MIC, respectively. (A) Western blotting to detect DGKζ protein with antibodies against the C terminus (C) or the N terminus (N; specific to splice variant 2) of DGKζ (n = 6 from three mice per group). (B) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) to measure mRNA expression of DGKζ (n = 5 to 6 from three mice per group). (C) Measurement of protein synthesis rate as assessed by Western blotting of puromycin (puro)–labeled peptides (PLP) (n = 5 to 6 from three mice per group). (D and E) Western blotting to detect phosphorylated (P) and total (T) p70 [n = to 6 from three mice per group (D) and n = 3 to 4 from three to four mice per group (E)]. (F) Measurement of protein degradation rate as assessed by tyrosine release (n = 3 to 5 from three to five mice per group). (G) Western blotting to detect the indicated proteins (n = 5 to 8 from three to four mice per group). Values were expressed as means (+SEM in graphs). *P < 0.05 compared to sham within the same genotype, #P < 0.05 compared to WT within the same surgery (C, D, F, and G) or MIC+ (E), ·P < 0.05 compared to non-MG132 within the same genotype and surgery, Student’s t test (A to B) or two-way ANOVA (C to G). Ubi, ubiquitin.

Article Snippet: Antibodies and plasmid construct Rabbit anti-p70 S6k (#2708), anti-4EBP1 (#9644), anti–phospho-4EBP1 (Thr 36/47 ; #2855), anti-eIF4G (#2469), anti-eIF4E (#2067), anti-eIF2α (#5324), anti-eEF2 (#2332), anti–pan-actin (#8456), anti–phospho-Akt (Thr 308 ; #9275), anti-Akt (#9272), anti-FoxO3a (#2497), anti-HA (#3724), anti-GFP (#2555), and anti-LDHA (#3558) antibodies were purchased from Cell Signaling.

Techniques: Control, Muscles, Western Blot, Variant Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Labeling, Ubiquitin Proteomics

Journal: Cellular reprogramming

Article Title: mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells.

doi: 10.1089/cell.2010.0011

Figure Lengend Snippet: FIG. 2. H7 hESCs show reduced levels of p70 S6K signaling compared to their differentiated progeny. (A) Representative Western blots from four separate experiments indicating decreased p70 S6K activation in hESCs compared to differentiated, fibroblast- like cells. (B) Representative images showing reduced S6 phosphorylation in H7 hESCs compared to H7 Diffs. S6 phosphorylation appears to be increased along the outer edges of hESC colonies. Scale bar, 40 mm.

Article Snippet: The p70 S6K constructs were generously donated to Addgene by Dr. John Blenis, Addgene catalog number 8989 (p70 S6K CON) and 8984 (p70 S6K WT).

Techniques: Western Blot, Activation Assay, Phospho-proteomics

Journal: Cellular reprogramming

Article Title: mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells.

doi: 10.1089/cell.2010.0011

Figure Lengend Snippet: FIG. 3. Suppression of mTORC1-mediated translation does not induce cell death or disrupt pluripotency in H7 hESCs. (A) Rapamycin treatment causes cell death in differentiated, but not undifferentiated cells. Representative images of untreated and treated H7 hESCs and H7 Diffs are shown. Scale bar, 100 mm. (B) Neither treatment with rapamycin nor siRNA against p70 S6K impairs colony formation or affects expression of pluripotency markers in H7 hESCs. Representative images for hESCs treated for 3 days with vehicle control or 20 nM rapamycin are shown. Also shown are representative images from H7 hESCs nucleofected with either control, nonspecific siRNA, or siRNA directed against p70 S6K. Typically, hESC colonies are smaller post nucleofection due to the requirement of single-cell suspensions. Staining for Oct4 and Nanog pluripotency markers are shown. Scale bar, 100 mM. Western blot insert confirms p70 S6K knockdown in hESCs with Actin serving as a loading control.

Article Snippet: The p70 S6K constructs were generously donated to Addgene by Dr. John Blenis, Addgene catalog number 8989 (p70 S6K CON) and 8984 (p70 S6K WT).

Techniques: Expressing, Control, Staining, Western Blot, Knockdown

Journal: Cellular reprogramming

Article Title: mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells.

doi: 10.1089/cell.2010.0011

Figure Lengend Snippet: FIG. 5. Expression of constitutively active p70 S6K alters cell morphology and causes a loss of Oct4 expression in H7 hESCs. Constitutively active p70 S6K induces differentia- tion of H7 hESCs. (A) Representative fluorescent imaging of H7 hESC nucleofected with GFP, GFP þ p70 S6K WT, or GFP þ p70 S6K CON and stained for Oct4. Arrows indicate nucleofected cells. Scale bar, 50 mm. (B) Graphical analysis of the percentage of GFP þ cells expressing Oct4. For each condition, approximately 300 cells were analyzed. Statistical significance, p < 0.01.

Article Snippet: The p70 S6K constructs were generously donated to Addgene by Dr. John Blenis, Addgene catalog number 8989 (p70 S6K CON) and 8984 (p70 S6K WT).

Techniques: Expressing, Imaging, Staining

Journal: Cellular reprogramming

Article Title: mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells.

doi: 10.1089/cell.2010.0011

Figure Lengend Snippet: FIG. 6. Knockdown of Rictor and TSC2 expression elevates p70 S6K activation and alters hESC morphology. Activation of p70 S6K by Rictor and TSC2 siRNA-mediated knockdown leads to changes in cell mor- phology to a more differentiated pheno- type. (A) Representative Western blots from multiple experiments showing ex- pression of proteins associated with mTORC1 (mammalian target of rapamycin complex 1) and mTORC2 signaling in H7 hESCs and H7 Diffs. (B) Western blot of immunoprecipitations of mTOR or TSC1 probed for Raptor, Rictor, or TSC2, re- spectively, in H7 hESCs and H7 Diffs. (C) Representative phase contrast images of H7 hESCs nucleofected with either control siRNA or Rictor=TSC2 siRNA mixture. Scale bar, 250 mm. (D) Western blot analysis confirming knockdown of TSC2 and Rictor as well as expression of p70 S6K, activated p70 S6K (p-T389), Oct4, and Actin.

Article Snippet: The p70 S6K constructs were generously donated to Addgene by Dr. John Blenis, Addgene catalog number 8989 (p70 S6K CON) and 8984 (p70 S6K WT).

Techniques: Knockdown, Expressing, Activation Assay, Western Blot, Control

Journal: Cellular reprogramming

Article Title: mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells.

doi: 10.1089/cell.2010.0011

Figure Lengend Snippet: FIG. 7. Diagram depicting mTORC signal- ing in hESCs and differentiated cells. In pluripotent hESCs (left panel), mTOR protein is mostly associated in mTORC2 (mTOR- Rictor), whereas mTORC1 (mTOR-Raptor) activity is suppressed by the heterodimer TSC1=TSC2. In differentiated cells (right panel), mTOR is highly associated with Raptor over Rictor leading to elevated p70 S6K activity. Increased protein translation, in turn, induces differentiation.

Article Snippet: The p70 S6K constructs were generously donated to Addgene by Dr. John Blenis, Addgene catalog number 8989 (p70 S6K CON) and 8984 (p70 S6K WT).

Techniques: Activity Assay