Journal: eLife

Article Title: Hyperactivity of mTORC1- and mTORC2-dependent signaling mediates epilepsy downstream of somatic PTEN loss

doi: 10.7554/eLife.91323

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit monoclonal anti-pS6 (S240/244) , Cell Signaling , Cat #5364 , 1:1000.

Techniques: Recombinant, Staining, Software

Journal: PLoS ONE

Article Title: FMRP S499 Is Phosphorylated Independent of mTORC1-S6K1 Activity

doi: 10.1371/journal.pone.0096956

Figure Lengend Snippet: ( A ) N2a cells were treated with vehicle 1 (DMSO), vehicle 2 (ethanol), rapamycin (Rapa), PF-4708671 (PF), bisindolylmaleimide V (B5) or okadaic acid (OA) for 1 hour prior to cell lysis followed by immunoblotting for pFMRP, pS6K1, pS6, pERK T202/Y204 (readout for OA), and their total protein counterparts. Laddered bracket to right of tFMRP indicates FMRP isoforms and the asterisk denotes a nonspecific band. S6K1 isoforms are marked by p80 and p75. ( B and C ) Statistical verification of stable pFMRP:tFMRP across all conditions despite a significant decrease in pS6:S6 subsequent to mTORC1-S6K1 inhibition (B) and increase in pERK:ERK subsequent to PP2a inhibition (C). One way ANOVA with post-hoc Dunnett’s test (N = 4 per condition. Error bars = SEM). ( D ) Model of pathway and effect of pharmacological inhibitor. ( E ) N2a cells. Immunoblotting for pFMRP, tFMRP and mTORC1 pathway components from N2a cells maintained in 5% serum, transferred to increasing concentrations of serum (0, 2.5, 5 or 10%), and treated with vehicle (DMSO), rapamycin (Rapa) or PF-4708671 (PF) for 24 hours. Laddered bracket to the right of tFMRP blot indicates FMRP isoforms and the asterisk a nonspecific band. S6K1 isoforms are indicated by p85 and p70. The asterisk to the right of the total S6K1 blot indicates residual tFMRP signal from the blot above. ( F ) Bar graphs of (E). Statistical analysis: unmatched two-way ANOVA corrected for multiple comparisons with a post-hoc Tukey’s test. N = 4 per condition. Error bars = SEM.

Article Snippet: pS6 S240/244 , Cell Signal (Rb) #5364 , 1∶10,000;20,000 , 5% BSA/TBST (total S6 must be probedfor first as pS6 is not efficiently stripped).

Techniques: Lysis, Western Blot, Inhibition

Journal: PLoS ONE

Article Title: FMRP S499 Is Phosphorylated Independent of mTORC1-S6K1 Activity

doi: 10.1371/journal.pone.0096956

Figure Lengend Snippet: ( A ) Cre expression and genetic recombination were verified by region-specific tdTomato expression (note tdTomato in telencephalon, Tel, but not cerebellum, Cb). Genotypes were confirmed by PCR: wild type mice (WT) have two wild type alleles (wt, 295bp amplicon), heterozygous mice (Het) have one wt and one mutant allele (mut, 370bp amplicon), and conditional mice have one floxed allele (fl, 480bp amplicon) with either a wt or mut allele. Asterisks indicate nonspecific bands ( B ) Immunoblotting of cortical lysates from P7 Tsc1 mice of different genotypes listed under the blots. ( C ) There is no statistical difference in pFMRP:tFMRP among genotypes despite significantly increased pS6:S6 in Tsc1 fl/mut vs. Tsc1 WT . pFMRP:tFMRP was quantified by unpaired, one-way ANOVA. pS6:S6 was quantified using unpaired, nonparametric one-sided Mann-Whitney test (** = p<0.01). Error bars STDEV. N = 3. ( D ) Model of serum-mediated mTORC1 pathway activation. ( E ) Immunoblots for pFMRP, pmTOR, pS6K1 and pS6 suggest that although the mTORC1 pathway responds to serum, pFMRP is not altered. N2a were cells maintained in 5% serum were transferred to 0, 2.5, 5 or 10% serum 1 hour prior to lysis. ( F ) Bar graphs of the phospho:total protein ratio shown in (E) showing that pFMRP:tFMRP is unchanged despite significantly increased mTORC1 pathway activity. Unpaired, nonparametric one-sided Mann-Whitney tests compared 0 and 10% serum conditions for each phospho-protein. We used a one-sided test because increased mTORC1 pathway activity is expected following serum application. Error bars = SEM. N = 4 per condition. ( G ) Model of mTORC1 pathway activation by overexpression. ( H ) Immunoblotting verified overexpression of transfected genes with the exception of TSC2 DN whose large C- and N-terminal deletions render it unrecognizable to many antibodies. N2a cell lysates were collected 48 hours post-transfection. ( I ) Immunoblotting for pFMRP, pS6K1, pS6 and their total protein counterparts. ( J ) Quantification of phospho:total protein ratios normalized to mock transfected cells – unpaired, one-way ANOVA corrected for multiple comparisons. P values were derived from post-hoc Dunnett’s test (*, **, *** & **** = P≤0.05, 0.01, 0.001 and 0.0001 respectively). N = 4 per condition.

Article Snippet: pS6 S240/244 , Cell Signal (Rb) #5364 , 1∶10,000;20,000 , 5% BSA/TBST (total S6 must be probedfor first as pS6 is not efficiently stripped).

Techniques: Expressing, Amplification, Mutagenesis, Western Blot, MANN-WHITNEY, Activation Assay, Lysis, Activity Assay, Over Expression, Transfection, Derivative Assay

Journal: PLoS ONE

Article Title: FMRP S499 Is Phosphorylated Independent of mTORC1-S6K1 Activity

doi: 10.1371/journal.pone.0096956

Figure Lengend Snippet: ( A ) CD1 mice were intraperitoneally injected (IP) with PF (75 mg/kg) and sacrificed at various time points thereafter. Hyperphosphorylation of S6K1 T389 (pS6K1) detected at 2 hours suggests that this compound can cross the blood brain barrier. N = 3 per time point. ( B ) Immunoblots from cortical lysates from CD1 mice treated IP with rapamycin (Rapa, 1.5 mg/kg for 5 days), PF-4708671 (PF, 75 mg/kg for 2 hours), and vehicle (DMSO) alone. Asterisks indicate nonspecific bands, the arrow indicates the pFMRP isoform, laddered bracket indicates the tFMRP isoforms, and S6K1 isoforms are indicated by p85 and p70. ( C ) Quantification verifies a significant decrease in pS6:S6 but no change in pFMRP:FMRP following mTORC1 or S6K1 inhibition in vivo . * P <0.05 and ** P <0.01 by unpaired, one-sided Mann-Whitney Test. A one-sided test was used considering that decreased pS6 levels were expected. N = 6 per condition. Error bars = SEM.

Article Snippet: pS6 S240/244 , Cell Signal (Rb) #5364 , 1∶10,000;20,000 , 5% BSA/TBST (total S6 must be probedfor first as pS6 is not efficiently stripped).

Techniques: Injection, Western Blot, Inhibition, In Vivo, MANN-WHITNEY

Journal: PLoS ONE

Article Title: FMRP S499 Is Phosphorylated Independent of mTORC1-S6K1 Activity

doi: 10.1371/journal.pone.0096956

Figure Lengend Snippet: List of antibodies.

Article Snippet: pS6 S240/244 , Cell Signal (Rb) #5364 , 1∶10,000;20,000 , 5% BSA/TBST (total S6 must be probedfor first as pS6 is not efficiently stripped).

Techniques: Blocking Assay

Journal: Cell Reports

Article Title: mTOR Signaling and SREBP Activity Increase FADS2 Expression and Can Activate Sapienate Biosynthesis

doi: 10.1016/j.celrep.2020.107806

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-pS6(S240/S244) , Cell Signaling Technology , Cat# 2215, RRID: AB_331682.

Techniques: Recombinant, cDNA Synthesis, SYBR Green Assay, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Software

Journal: Cancers

Article Title: A Phase Ib Study of Sotrastaurin, a PKC Inhibitor, and Alpelisib, a PI3Kα Inhibitor, in Patients with Metastatic Uveal Melanoma.

doi: 10.3390/cancers13215504

Figure Lengend Snippet: Figure 3. Western blot analysis of PKC and PI3Kα-AKT-mTOR pathway inhibition. Paired tumor biopsies were assessed by Western blot for pAKT, pS6, pERK1/2, pMARCKS, and the respective total proteins. GAPDH was used as a loading control. Clinical outcomes for these respective patients are listed. Protein quantitation of the pre-treatment (left) and post-treatment (right) Western blot analysis performed with Image J. The pre-treatment sample expression level represents a baseline of 100%, with the post-treatment sample expression levels relative to this baseline. Bar plots represent pAKT/total AKT, pS6/total S6, pERK1/2/ total ERK1/2, and pMARCKS/MARCKS, respectively.

Article Snippet: Antibodies used to probe were: pAKT (Ser473, #4060, Clone D9E), Pan AKT (#2920, Clone 40D4), pS6 (S240/244, #4858, Clone D57.2.2E), S6 ribosomal protein (Ser235/236, #2317, Clone 54D2), pMARCKS (Ser152/156, #2741), MARCKS (#5607, Clone D88D11), GAPDH (#5174, Clone D16HI1), pERK1/2 (Y204, #4370, Clone), ERK1/2 (#4695, Clone 137F5), Cyclin D1(#2978, Clone 92G2), Cyclin E1 (#20808, Clone D7T3U), Cyclin A2 (#67955, Clone E6D1J), Bcl-2 (#4223, Clone D55G8), and GLUT4 (Santa Cruz Biotech #SC-53566, Clone IF8), obtained from Cell Signaling Technology Inc., unless otherwise noted.

Techniques: Western Blot, Inhibition, Control, Protein Quantitation, Expressing