mouse anti tsc2  (Novus Biologicals)

Journal: Journal of neuroinflammation

Article Title: Microglial SIRT1 activation attenuates synapse loss in retinal inner plexiform layer via mTORC1 inhibition.

doi: 10.1186/s12974-023-02886-8

Figure Lengend Snippet: Fig. 3 SIRT1 mediates the deacetylation of TSC2 at lysine residues 1473. A Exogenous TSC2 was targeted by acetylation in HEK293 cells. Shown is immunoblot (IB) analysis of whole-cell lysates (input) and anti-Flag immunoprecipitates derived from HEK293 cells transfected with the indicated constructs. B Endogenous TSC2 was targeted by acetylation in BV2 cells. Shown is IB analysis of input and anti-TSC2 immunoprecipitates derived from BV2 microglia cells. IgG IP served as control. C Sirt1 promoted TSC2 deacetylation in cells. Shown is IB analysis of input and anti-Flag immunoprecipitates derived from HEK293 cells transfected with Flag-TSC2 along with hemagglutinin (HA)-tagged constructs. HA-SIRT1-HY, catalytically inactive SIRT1. D Treatment with EX527, Sirt1 specific inhibitor, could reduce TSC2 acetylation. Shown is IB analysis of input and anti-Flag immunoprecipitates derived from HEK293 cells transfected with Flag-TSC2 along with no another treatment, DMSO treatment and EX527 treatment, respectively. E, F Exogenous interaction between TSC2 and Sirt1. Shown is IB analysis of input and anti-Flag (E) or anti HA (F) immunoprecipitates derived from HEK293 cells transfected with Flag-TSC2 along with hemagglutinin (HA)-tagged constructs or transfected with HA-Sirt1 along with Flag-tagged constructs. IgG IP served as control. G, H Endogenous interaction between TSC2 and Sirt1. Shown is IB analysis of input and anti-TSC2 (G) or anti-Sirt1 (H) immunoprecipitates derived from BV2 cells. IgG IP served as control. I Representative immunofluorescence images showing colocalization of HA-Sirt1 (green) and TSC2 (red) in BV2 cells. Scale bar, 40 μm. J Acetylation-deficient K1473R significantly diminished TSC2 acetylation. Shown is IB analysis of input and anti-Flag immunoprecipitates derived from HEK293 cells transfected with Flag-TSC2. K Acetylation-deficient TSC2 K1473R mutant markedly diminished the interaction between Sirt1 and TSC2. Shown is IB analysis of input and anti-HA immunoprecipitates derived from HEK293 cells transfected with HA-Sirt1 along with Flag-tagged constructs

Article Snippet: The following antibodies were used in western blot: anti-TSC2 antibody (#4308; cell signaling technology; 1:1000); anti-HA-Tag antibody (#sc-7392; Santa Cruz; 1:100); anti-Sirt1 antibody (ab189494; Abcam; 1:1000); anti-phospho-tuberin/ TSC2 (Ser1387) antibody (#5584; cell signaling technology; 1:1000); anti-FLAG-Tag antibody (#8146; cell signaling technology; 1:1000); anti-p70 S6 kinase antibody (#2708; cell signaling technology; 1:1000); anti-phosphop70 S6 kinase antibody (#9234; cell signaling technology; 1:1000).

Techniques: Western Blot, Derivative Assay, Transfection, Construct, Control, Immunofluorescence, Mutagenesis

Journal: Journal of neuroinflammation

Article Title: Microglial SIRT1 activation attenuates synapse loss in retinal inner plexiform layer via mTORC1 inhibition.

doi: 10.1186/s12974-023-02886-8

Figure Lengend Snippet: Fig. 4 SIRT1-mediated TSC2 deacetylation decreases TSC2 phosphorylation at tyrosine 1462. A TSC2 acetylation was related to its phosphorylation. When compared with wildtype TSC2, phosphorylation level increased in acetylation-mimetic TSC2 K1473Q mutant while decreased in acetylation-deficient TSC2 K1473R mutant. Shown is IB analysis of whole-cell lysates derived from HEK293 cells transfected with Flag-tagged constructs. B Ratio of the p-TSC2 band intensities to Flag band intensities in A. *P < 0.05; **P < 0.01. N = 3. C Overexpression of Sirt1 reduced TSC2 phosphorylation. Shown is IB analysis of whole-cell lysates derived from HEK293 cells transfected with Flag-TSC2 along with HA-tagged constructs. D Ratio of the p-TSC2 band intensities to Flag band intensities in C. ***P < 0.001; ns, P > 0.05. N = 3. E Inhibition of Sirt1 activity via EX527 increased TSC2 phosphorylation level. Shown is IB analysis of whole-cell lysates derived from HEK293 cells transfected with Flag-TSC2 along with or without EX527 treatment. F Ratio of the p-TSC2 band intensities to Flag band intensities in E. ***P < 0.001. N = 3. G Sirt1 overexpression could attenuate TSC2 phosphorylation level induced by LPS plus IFN-γ treatment. Shown is IB analysis of whole-cell lysates derived from BV2 cells under different intervention. H Ratio of the p-TSC2 band intensities to Flag band intensities in E. **P < 0.01. N = 3. I EX527 induced inhibition of Sirt1 activity could further enhance TSC2 phosphorylation level induced by LPS plus IFN-γ treatment. Shown is IB analysis of whole-cell lysates derived from BV2 cells under different intervention. J Ratio of the p-TSC2 band intensities to Flag band intensities in I. *P < 0.05; **P < 0.01. N = 3. K Schematic drawing of the interaction of Sirt1, TSC2 and mTOR. SIRT1 deacetylated TSC2 at lysine residues 1473, further reduced its phosphorylation level of TSC2 at tyrosine 1462 and finally activated TSC2 to inhibit mTOR

Article Snippet: The following antibodies were used in western blot: anti-TSC2 antibody (#4308; cell signaling technology; 1:1000); anti-HA-Tag antibody (#sc-7392; Santa Cruz; 1:100); anti-Sirt1 antibody (ab189494; Abcam; 1:1000); anti-phospho-tuberin/ TSC2 (Ser1387) antibody (#5584; cell signaling technology; 1:1000); anti-FLAG-Tag antibody (#8146; cell signaling technology; 1:1000); anti-p70 S6 kinase antibody (#2708; cell signaling technology; 1:1000); anti-phosphop70 S6 kinase antibody (#9234; cell signaling technology; 1:1000).

Techniques: Phospho-proteomics, Mutagenesis, Derivative Assay, Transfection, Construct, Over Expression, Inhibition, Activity Assay

Journal: Journal of neuroinflammation

Article Title: Microglial SIRT1 activation attenuates synapse loss in retinal inner plexiform layer via mTORC1 inhibition.

doi: 10.1186/s12974-023-02886-8

Figure Lengend Snippet: Fig. 7 A schematic diagram of proposed crosstalk among Sirt1, TSC2 and mTORC1 in regulation of microglial activation. Microglial activation mediated synapse loss occurs in Retinal IPL after optic nerve injury. In the regulation process of microglial activation, Sirt1 enhances TSC2 activity via deacetylating it at lysine residues 1473 and further reducing its phosphorylation level at tyrosine 1462. Activated TSC2 exerts the inhibition effect on mTORC1. Hence, through the above signaling pathway, microglial Sirt1 could attenuate RGCs injury via inhibiting microglial activation

Article Snippet: The following antibodies were used in western blot: anti-TSC2 antibody (#4308; cell signaling technology; 1:1000); anti-HA-Tag antibody (#sc-7392; Santa Cruz; 1:100); anti-Sirt1 antibody (ab189494; Abcam; 1:1000); anti-phospho-tuberin/ TSC2 (Ser1387) antibody (#5584; cell signaling technology; 1:1000); anti-FLAG-Tag antibody (#8146; cell signaling technology; 1:1000); anti-p70 S6 kinase antibody (#2708; cell signaling technology; 1:1000); anti-phosphop70 S6 kinase antibody (#9234; cell signaling technology; 1:1000).

Techniques: Activation Assay, Activity Assay, Phospho-proteomics, Inhibition

Journal: Life Science Alliance

Article Title: Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt

doi: 10.26508/lsa.202101169

Figure Lengend Snippet: (A) Example immunoblots and summary data of dose dependent decline in ET-1 induced S6K and RSK2 phosphorylation by ERK1/2 inhibition (SCH) in NRVMs. Concomitant ERK1/2 phosphorylation itself did not decline, whereas Akt phosphorylation rose with increasing SCH dose (0.01–10 μM). N = 6/group; **** P < 1 × 10 −4 , *** P ≤ 0.0006, ** P ≤ 0.006, * P = 0.01; Welch one-way ANOVA, Dunnett’s multiple comparisons test (MCT). (B) Inhibition of Akt does not significantly reduce p/t S6K stimulated by ET-1. N = 6/group; Kruskal–Wallis test P < 0.0007 for each; Dunn’s MCT: ** P = 0.002; * P < 0.026. (C) Expression of TSC2 S1364E (SE) reduces ET-1–stimulated S6K in a dose-dependent manner; ERK1/2 activity is unchanged, and Akt activity rises with higher SE expression. N = 4/group; one-way ANOVA ( P ≤ 3 × 10 −10 for each protein), Sidak’s MCT: **** P < 10 −5 ; *** P < 0.0002. Source data are available for this figure.

Article Snippet: Primary antibodies were targeted to: Total TSC2 #4308, phospho-p70 S6 kinase (Thr389) #9205, total p70 S6 kinase #9202, phospho-4E-BP1 (Ser65) #9451 and total 4E-BP1 #9452, phospho-Akt (Ser473) #9271, phospho-Akt (Thr308) #13038, and total Akt #9272, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) #9101 and total p44/42 MAPK (ERK1/2) #9102, pRSK (T359/S363) #9344 and total RSK #9355, pRSK2 S227 #3556S and total RSK2 (#5528S) (all from Cell Signaling Technology, and used a 1:1,000 dilution), and p-TSC2 S1365 (mouse) #120718 (NovoPro Labs, 1:500).

Techniques: Western Blot, Inhibition, Expressing, Activity Assay

Journal: Life Science Alliance

Article Title: Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt

doi: 10.26508/lsa.202101169

Figure Lengend Snippet: (A) Effect of ERK-1/2 inhibitor U0216 on ET-1–stimulated response. NRVMs were stimulated with ET-1 in presence or absence of ERK-1/2 inhibitor U0216. (n = 6/group). Summary analyzed by Welch one-way ANOVA (variance differences between groups) and multiple comparisons Dunnett’s test P -values displayed. (B) Effect of TSC2S S1364E (SE) expression on RSK2 activation. NRVMs infected with WT or SE form of TSC2, exposed to ET-1 ± ERK1/2 inhibitors. (n = 6/group). Summary data analyzed by one-way-ANOVA, Dunnett’s multiple comparisons test P -values shown.

Article Snippet: Primary antibodies were targeted to: Total TSC2 #4308, phospho-p70 S6 kinase (Thr389) #9205, total p70 S6 kinase #9202, phospho-4E-BP1 (Ser65) #9451 and total 4E-BP1 #9452, phospho-Akt (Ser473) #9271, phospho-Akt (Thr308) #13038, and total Akt #9272, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) #9101 and total p44/42 MAPK (ERK1/2) #9102, pRSK (T359/S363) #9344 and total RSK #9355, pRSK2 S227 #3556S and total RSK2 (#5528S) (all from Cell Signaling Technology, and used a 1:1,000 dilution), and p-TSC2 S1365 (mouse) #120718 (NovoPro Labs, 1:500).

Techniques: Expressing, Activation Assay, Infection

Journal: Life Science Alliance

Article Title: Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt

doi: 10.26508/lsa.202101169

Figure Lengend Snippet: (A, B) Example immunoblots and (B) summary data showing ET-1–stimulated S6K in NRVMs is augmented by expression of TSC2 S1364A (SA) without altering ERK1/2 phosphorylation. This stimulation is blocked by ERK1/2 (SCH) or mTOR (rapamycin, Rapa) inhibition. N = 4/group; P -values for two-way ANOVA for genotype and interaction of genotype and condition. **** P < 0.0001 by Sidak’s MCT. (C) Effect of ET-1 stimulation on S6K and ERK1/2 phosphorylation in NRMVs co-expressing TSC2 S1364A and PKG1α C42S and then exposed to vehicle (Veh), PDE5 (P5-i) (1 μM), or PDE9 (P9-i) (0.1 μM) inhibition, or soluble guanylate cyclase activation (GC-1) (0.1 μM). N = 6/group; Welch one-way ANOVA, Dunnett’s MCT; *** P ≤ 0.001; **** P = 0.00006. (D) Combined analysis of data from and bi-normalized to span from 1.0 with vehicle control to 4.0 for peak p/t S6K response. Data show strong correlation between p/t S6K and p/t ERK1/2 but not p/t Akt. P -values are for linear regression of each respective relation. Source data are available for this figure.

Article Snippet: Primary antibodies were targeted to: Total TSC2 #4308, phospho-p70 S6 kinase (Thr389) #9205, total p70 S6 kinase #9202, phospho-4E-BP1 (Ser65) #9451 and total 4E-BP1 #9452, phospho-Akt (Ser473) #9271, phospho-Akt (Thr308) #13038, and total Akt #9272, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) #9101 and total p44/42 MAPK (ERK1/2) #9102, pRSK (T359/S363) #9344 and total RSK #9355, pRSK2 S227 #3556S and total RSK2 (#5528S) (all from Cell Signaling Technology, and used a 1:1,000 dilution), and p-TSC2 S1365 (mouse) #120718 (NovoPro Labs, 1:500).

Techniques: Western Blot, Expressing, Inhibition, Activation Assay

Journal: Life Science Alliance

Article Title: Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt

doi: 10.26508/lsa.202101169

Figure Lengend Snippet: (A) Insulin-stimulated pS6K in MEFs is not altered by ERK1/2 inhibition but is by Akt inhibition; example immunoblot (left) and summary data (right) shown. N = 6/group; one-way ANOVA, Holm–Sidak MCT: ** P ≤ 0.004, *** P = 0.0002; ****P ≤ 2 × 10 −5 . (B) Example immunoblot and summary results for insulin stimulation in TSC2 KO MEFs infected with AdV expressing empty vector (KO), or WT, SA, or SE TSC2. N = 12/group; two-Way ANOVA, Sidak’s MCT; *P < 2 × 10 −7 versus Vehicle KO; †P < 3 × 10 −7 versus KO+ insulin; ‡P ≤ 6 × 10 −7 versus KO+ insulin. (C) Insulin-stimulated S6K is fully blocked by rapamycin (Rapa) or torkinib (Tork), contrasting to effect from SE mutant. N = 6/group; Welch one-way ANOVA, Dunnett’s test; * P < 0.0003 versus other groups. (D) PDGF stimulation potently activates Akt and S6K, and this response is not significantly altered by TSC2 mutants versus WT. N = 6/group; two-way ANOVA, P = 3 × 10 −12 for PDGF effect, 0.7 for genotype effect, and 0.8 for genotype–PDGF interaction. Source data are available for this figure.

Article Snippet: Primary antibodies were targeted to: Total TSC2 #4308, phospho-p70 S6 kinase (Thr389) #9205, total p70 S6 kinase #9202, phospho-4E-BP1 (Ser65) #9451 and total 4E-BP1 #9452, phospho-Akt (Ser473) #9271, phospho-Akt (Thr308) #13038, and total Akt #9272, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) #9101 and total p44/42 MAPK (ERK1/2) #9102, pRSK (T359/S363) #9344 and total RSK #9355, pRSK2 S227 #3556S and total RSK2 (#5528S) (all from Cell Signaling Technology, and used a 1:1,000 dilution), and p-TSC2 S1365 (mouse) #120718 (NovoPro Labs, 1:500).

Techniques: Inhibition, Western Blot, Infection, Expressing, Plasmid Preparation, Mutagenesis

Journal: Life Science Alliance

Article Title: Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt

doi: 10.26508/lsa.202101169

Figure Lengend Snippet: (A) Insulin did not alter 4E-BP1 in KO MEFs, but increased in cells in which TSC2 was re-expressed ( P = 1.5 × 10 −5 for insulin effect in WT, SE, SA group ± insulin two-way ANOVA; n = 12/group). P -values are for Sidak’s multiple-comparisons test comparing results ± insulin within TSC2 genotype group. (B) PDGF stimulation has no effect on 4E-BP1 phosphorylation independent of the TSC2 genotype expressed in TSC2-KO MEFs. (n = 6/group). Analyzed by two-way ANOVA. (C) 4E-BP1 phosphorylation declines markedly and similarly in NRVMs subjected to simulated ischemia (SI). (n = 4/group). Two-way ANOVA, genotype interaction P = 0.44; SI effect 6 × 10 −10 ; Sidak’s multiple comparisons test: *P = 6 × 10 −5 Si versus control. (D) 4E-BP1 phosphorylation increases with amino acid (AA) repletion markedly and similarly in NRVMs. (n = 8/group). Two-way ANOVA, genotype interaction P = 0.71; AA repletion effect 6 × 10 −10 ; Sidak’s multiple comparisons test: *P ≤ 8 × 10 −5 † P = 0.001 for AA deplete versus AA replete.

Article Snippet: Primary antibodies were targeted to: Total TSC2 #4308, phospho-p70 S6 kinase (Thr389) #9205, total p70 S6 kinase #9202, phospho-4E-BP1 (Ser65) #9451 and total 4E-BP1 #9452, phospho-Akt (Ser473) #9271, phospho-Akt (Thr308) #13038, and total Akt #9272, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) #9101 and total p44/42 MAPK (ERK1/2) #9102, pRSK (T359/S363) #9344 and total RSK #9355, pRSK2 S227 #3556S and total RSK2 (#5528S) (all from Cell Signaling Technology, and used a 1:1,000 dilution), and p-TSC2 S1365 (mouse) #120718 (NovoPro Labs, 1:500).

Techniques:

Journal: Life Science Alliance

Article Title: Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt

doi: 10.26508/lsa.202101169

Figure Lengend Snippet: (A) Example immunoblot of MEFs exposed to thrombin showing balanced activation of ERK1/2 and Akt. (B) Example immunoblot and summary data for TSC2 KO MEFs and cells with WT TSC2 re-expressed, exposed to thrombin ± Akt inhibition (MK2206). N = 4/group TSC2-KO; N = 11/group WT. P -values displayed are for Kruskal–Wallis test, **** P = .00009, ** P = 0.009 by Dunn’s MCT. (C) Example immunoblot and summary data for the same experiment but ± ERK1/2 inhibitor (SCH). N = 6/group; KW test P -values displayed; Dunn’s MCT: † P = 0.1, ** P = 0.007. (D) Example immunoblot for KO MEFs re-expressing either TSC2-WT or TSC2 S1364E , stimulated with thrombin ± Akt inhibitor (MK2206). (E) Percent rise in p/t S6K due to thrombin in WT versus SE TSC2 expressing KO MEFs. N = 7–8/group; P -value displayed Mann–Whitney U test. (F) Effect of Akt inhibition on p/t S6K response to thrombin in TSC2-KO MEFs expressing WT or SE TSC2. N = 8/group; two-way ANOVA, Sidak’s MCT— P -values displayed. (G) Effect of ERK1/2 inhibition by SCH772984 on p/t S6K in TSC2-KO MEFs expressing WT or TSC2 S1364E . two-way ANOVA with Sidak’s MCT P -values shown. † P = 0.04 versus WT; * P = 0.022 versus SE with vehicle controls. (H) Differential effect of ET1 or insulin stimulation on TSC2 phosphorylation at S1364 N = 6/group; one-way ANOVA, Sidak’s MCT. * P = 0.012; ** P = 0.001; **** P = 0.00008. Source data are available for this figure.

Article Snippet: Primary antibodies were targeted to: Total TSC2 #4308, phospho-p70 S6 kinase (Thr389) #9205, total p70 S6 kinase #9202, phospho-4E-BP1 (Ser65) #9451 and total 4E-BP1 #9452, phospho-Akt (Ser473) #9271, phospho-Akt (Thr308) #13038, and total Akt #9272, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) #9101 and total p44/42 MAPK (ERK1/2) #9102, pRSK (T359/S363) #9344 and total RSK #9355, pRSK2 S227 #3556S and total RSK2 (#5528S) (all from Cell Signaling Technology, and used a 1:1,000 dilution), and p-TSC2 S1365 (mouse) #120718 (NovoPro Labs, 1:500).

Techniques: Western Blot, Activation Assay, Inhibition, Expressing, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt

doi: 10.26508/lsa.202101169

Figure Lengend Snippet: (A) NRMVs exposed to simulated ischemia (SI) display marked suppression of S6K activation that is similar regardless of the TSC2 S1345 genotype. There is slightly less activation after SI in TSC2 S1364A expressing cells. N = 4/group; Kruskal–Wallis test, Dunn’s MCT: * P = 0.03 versus WT-SI. (B) Effect of serum depletion on S6K activation. p/t S6K is significantly greater in SA expressing cells, but all decline with serum depletion independent of TSC2 genotype. N = 6–7/group; two-way ANOVA P -values provided; Sidak’s MCT: P -values shown. (C) Amino acid (AA) repletion increases p/t S6K similarly independent of the TSC2 genotype expressed. P -values are for two-way ANOVA genotype effect. Effect of genotype: P = 0.72; Genotype × AA repletion, P = 0.44. Source data are available for this figure.

Article Snippet: Primary antibodies were targeted to: Total TSC2 #4308, phospho-p70 S6 kinase (Thr389) #9205, total p70 S6 kinase #9202, phospho-4E-BP1 (Ser65) #9451 and total 4E-BP1 #9452, phospho-Akt (Ser473) #9271, phospho-Akt (Thr308) #13038, and total Akt #9272, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) #9101 and total p44/42 MAPK (ERK1/2) #9102, pRSK (T359/S363) #9344 and total RSK #9355, pRSK2 S227 #3556S and total RSK2 (#5528S) (all from Cell Signaling Technology, and used a 1:1,000 dilution), and p-TSC2 S1365 (mouse) #120718 (NovoPro Labs, 1:500).

Techniques: Activation Assay, Expressing, Serum Depletion

Journal: Life Science Alliance

Article Title: Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt

doi: 10.26508/lsa.202101169

Figure Lengend Snippet: (A) Body weight increases similarly in homozygous knock-in mice with TSC2 S1364A , TSC2 S1364E , or littermate (WT) controls over an 18-wk duration of high fat diet (HFD). Results for control diet (ConD) at the later time point are also displayed. Group sizes provided in figure. Analysis by two-way ANOVA, P = 0.23 for time × genotype; P = 0.11 for genotype; P < 10 −10 for time effect. (B) Fasting blood glucose for each TSC2 genotype. Two-way ANOVA: P = 0.9 for genotype × diet interaction; HFD versus control diet: * P = 0.002, † P = 0.0003; ‡ P = 001. (C) Glucose response test for each group. N = 6/group; two-way ANOVA: P = 0.31 for time × genotype, 0.57 genotype, P = 10 −9 time. (D) Example H/E-stained liver histology from WT, TSC2 S1364A , and TSC2 S1364E KI mice with control versus HFD. There was similar marked hepatic steatosis regardless of the TSC2 genotype. Controls on standard diet are shown in the left column. Data representative of group results n = 6–8/group, with P > 0.6 for genotype effect on steatosis (χ 2 test). Source data are available for this figure.

Article Snippet: Primary antibodies were targeted to: Total TSC2 #4308, phospho-p70 S6 kinase (Thr389) #9205, total p70 S6 kinase #9202, phospho-4E-BP1 (Ser65) #9451 and total 4E-BP1 #9452, phospho-Akt (Ser473) #9271, phospho-Akt (Thr308) #13038, and total Akt #9272, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) #9101 and total p44/42 MAPK (ERK1/2) #9102, pRSK (T359/S363) #9344 and total RSK #9355, pRSK2 S227 #3556S and total RSK2 (#5528S) (all from Cell Signaling Technology, and used a 1:1,000 dilution), and p-TSC2 S1365 (mouse) #120718 (NovoPro Labs, 1:500).

Techniques: Knock-In, Staining

Journal: Life Science Alliance

Article Title: Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt

doi: 10.26508/lsa.202101169

Figure Lengend Snippet: A G q,11 – GPCR typified by ET-1 receptor and tyrosine kinase receptor typified by insulin receptor, and mixed receptor (e.g., thrombin) are depicted with their downstream signaling. ET-1 prominently activates PLCβ-PKC-ERK1/2 and Ras-ERK1/2 pathways leading TSC2 phosphorylation reducing its suppression of mTORC1 to increase p/t S6K. Insulin more prominently activates PI3K-Akt, phosphorylating TSC2 at different sites but also leading to mTORC1 activation. Thrombin engages both cascades. TSC2 S1364—modified by mutagenesis to block (S1364A, SA) or mimic (S1364E, SE) its phosphorylation—alters co-modulation of mTORC1 due to ERK1/2 but not to Akt signaling via TSC2. In vivo examples of this bias is found by marked impact of S1364 modulation on cardiac pressure overload as reported , but negligible effect on high-fat diet obesity and metabolic syndrome observed in the current study.

Article Snippet: Primary antibodies were targeted to: Total TSC2 #4308, phospho-p70 S6 kinase (Thr389) #9205, total p70 S6 kinase #9202, phospho-4E-BP1 (Ser65) #9451 and total 4E-BP1 #9452, phospho-Akt (Ser473) #9271, phospho-Akt (Thr308) #13038, and total Akt #9272, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) #9101 and total p44/42 MAPK (ERK1/2) #9102, pRSK (T359/S363) #9344 and total RSK #9355, pRSK2 S227 #3556S and total RSK2 (#5528S) (all from Cell Signaling Technology, and used a 1:1,000 dilution), and p-TSC2 S1365 (mouse) #120718 (NovoPro Labs, 1:500).

Techniques: Activation Assay, Modification, Mutagenesis, Blocking Assay, In Vivo

Journal: American Journal of Physiology - Cell Physiology

Article Title: Whole egg, but not egg white, ingestion induces mTOR colocalization with the lysosome after resistance exercise

doi: 10.1152/ajpcell.00225.2018

Figure Lengend Snippet: Tuberous sclerosis 2 (TSC2) and mechanistic target of rapamycin (mTOR) colocalization with Ras homolog enriched in brain (Rheb) in response to whole eggs or egg whites following resistance exercise. Immunofluorescence quantification of mTOR/TSC2 (red) and Rheb (green) colocalization, displayed as a composite image (merge) and WGA (blue) (n = 10/condition). Yellow/orange regions represent TSC2 and Rheb (A) mTOR and Rheb colocalization (C). Each panel represents one subject from whole eggs and egg whites across the experimental time course. Group data are quantified and reported as TSC2 and Rheb colocalization (B) and mTOR and Rheb colocalization (D). Open circles represent whole eggs and closed circles represent egg whites. All data are presented relative to Fasted. Scale bar represents 100-μm area. Data are presented as means ± SE and were analyzed using two-way repeated-measures ANOVA. *P < 0.01, different from Fasted in same condition. Immunofluorescence image was obtained on a Leica DMI8 confocal microscope of mTOR (red) and Rheb (green) interaction, displayed as a composite image (merge) and WGA (blue) (E). Sections were viewed at ×60 1.30 numerical aperture oil immersion objective to visualize the signal being primarily intracellular and/or within the fiber junctions. Scale bar represents 20-μm area (E).

Article Snippet: The mouse monoclonal anti-mTOR (no. 05-1592) antibody was purchased from Millipore (Toronto, ON, Canada) and mouse monoclonal anti-TSC2 (no. AM1919b-EV) was purchased from FroggaBio (Toronto, ON, Canada).

Techniques: Immunofluorescence, Microscopy