Journal: The Journal of Biological Chemistry

Article Title: Calmodulin enhances mTORC1 signaling by preventing TSC2-Rheb binding

doi: 10.1016/j.jbc.2024.108122

Figure Lengend Snippet: Ca 2+ /CaM prevents TSC2-Rheb interaction . A , the amino acids sequence for the CaM-binding region in TSC2 was shown, which matches the motif for the 1-10-14 type ( left ). The predicted structure of Rheb and the GAP domain of TSC2 using AlphaFold2 is shown ( right ). The parallel α-helices which are thought to support the binding of Rheb with TSC2 are shown in blue and red (residues 1716–1731). α-helix ( red ) corresponds to the CaM-binding region. B , schematic presentation of the HiBiT lytic assay. The assay was performed in three steps. Strep-GFP-tagged TSC2 (GFP-TSC2) and HiBiT-tagged Rheb (HiBiT-Rheb) were mixed in vitro (Step 1). HiBiT-Rheb bound to GFP-TSC2 were purified by immunoprecipitation with anti-GFP antibody (Step 2). NanoLuc luciferase activity can be recovered by reconstitution of HiBiT with LgBiT, and then generates luminescence using the substrate (Step 3). The amounts of Rheb in GFP-TSC2 immunoprecipitates were detected as the luminescence intensity. C , HEK293T cells were transiently transfected with the plasmid encoding Strep-GFP (GFP), Strep-GFP-TSC2 (GFP-TSC2), or HiBiT-Rheb. After 24 h of transfection, cells were lysed and respective lysates were mixed. Then, the cell lysates were subjected to immunoprecipitation using the anti-GFP antibody overnight, followed by the addition of Protein G magnetic beads. After washing the beads, the amounts of HiBiT and Strep were monitored as described in Experimental procedures. The ratio of HiBiT-Rheb to GFP or GFP-TSC2 was shown ( left ). The samples were also analyzed with Western blotting using the anti-GFP antibody and LgBiT blotting for detecting HiBiT-Rheb ( right ). D , HEK293 T cells were transiently transfected with the plasmid encoding Strep-GFP-TSC2 or HiBiT-Rheb. After 24 h of transfection, cells were lysed and respective cell lysates were mixed in the absence (Buffer) or presence of purified His-CaM (+His-CaM, 5.5 μM) together with 100 μM of CaCl 2 . Subsequent procedures were done as in (C). Graphs represent mean ± SD of five independent experiments, One-way ANOVA with Tukey’s test,∗∗∗ p < 0.001. E , assays were done as described in (D) using different amounts of His-CaM (0, 0.17, 0.25, 0.50, 1.0, 2.0, 4.0, 6.0 μM). Average results from two independent experiments were used to calculate the IC 50 value.

Article Snippet: For immunofluorescence, TSC2 (D93F12, Cell Signaling Technology #4308) and LAMP2 (H4B4, Proteintech 65053-1-Ig) were used as primary antibodies.

Techniques: Sequencing, Binding Assay, In Vitro, Purification, Immunoprecipitation, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Magnetic Beads, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Calmodulin enhances mTORC1 signaling by preventing TSC2-Rheb binding

doi: 10.1016/j.jbc.2024.108122

Figure Lengend Snippet: Ca 2+ is required for CaM to prevent TSC2-Rheb interaction . A , HEK293T cells were transiently transfected with the plasmid encoding Strep-GFP-TSC2 or HiBiT-Rheb. After 24 h of transfection, cells were lysed and respective cell lysates were mixed in the absence (−) or presence (+) of purified His-CaM (1 μM) together with 100 μM of CaCl 2 (Ca 2+ ) or 5 mM EGTA (EGTA). Subsequent procedures were done as in Fig. 1 C . Graphs represent mean ± SD of four independent experiments, One-way ANOVA with Tukey’s test, ∗∗ p < 0.01. B , HEK293T cells were transiently transfected with the plasmid encoding Strep-GFP-TSC2 or HiBiT-Rheb. After 24 h of transfection, cells were lysed and respective cell lysates were mixed in the absence (Ctrl) or presence of purified 1 μM or 5.5 μM of wild-type His-CaM (WT) or Ca 2+ binding-defective His-CaM (DA) together with 100 μM of CaCl 2 . Subsequent procedures were done as in (A). Graphs represent mean ± SD of three independent experiments, One-way ANOVA with Tukey’s test, ∗∗ p < 0.01, ∗∗∗ p < 0.001. C , The (B) samples were also analyzed with Western blotting using the anti-GFP and anti-6 × His antibody and LgBiT blotting for detecting HiBiT-Rheb. D , schematic diagram of (B). WT CaM prevents TSC2 and Rheb interaction, while Ca 2+ binding-defective CaM mutant (DA) does not interfere with the interaction.

Article Snippet: For immunofluorescence, TSC2 (D93F12, Cell Signaling Technology #4308) and LAMP2 (H4B4, Proteintech 65053-1-Ig) were used as primary antibodies.

Techniques: Transfection, Plasmid Preparation, Purification, Binding Assay, Western Blot, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Calmodulin enhances mTORC1 signaling by preventing TSC2-Rheb binding

doi: 10.1016/j.jbc.2024.108122

Figure Lengend Snippet: Binding of CaM to TSC2 contributes to the attenuation of TSC2-Rheb interaction . A , HEK293T TSC2 KO cells were transiently transfected with the plasmid encoding wild-type (WT), CaM-binding region deleted mutant (ΔCaM) of GFP-TSC2 or HiBiT-Rheb. After 24 h of transfection, cells were lysed and respective cell lysates were mixed. Then, the cell lysates were subjected to immunoprecipitation using the anti-GFP antibody for overnight, followed by the addition of Protein G magnetic beads. After washing the beads, the amounts of HiBiT and GFP were monitored as described in Experimental procedures. Graphs represent mean ± SD of three independent experiments, Two-tailed unpaired Student’s t test, ∗ p < 0.05. B , HEK293T TSC2 KO cells were transiently transfected with the plasmid encoding wild-type (WT), CaM-binding region deleted mutant (ΔCaM) of GFP-TSC2 or HiBiT-Rheb. After 24 h of transfection, cells were lysed and respective cell lysates were mixed in the absence (0 μM) or presence (0.5 μM, 1.0 μM) of purified His-CaM together with 100 μM of CaCl 2 . Subsequent procedures were done as in (A). Graphs represent mean ± SD of three independent experiments, Two-tailed unpaired Student’s t test, ∗ p < 0.05. C , HEK293T TSC2 KO cells were transiently transfected with the plasmid encoding wild-type (WT), CaM-binding region deleted mutant (ΔCaM) of GFP-TSC2. Cell lysates were subjected to immunoprecipitation (IP) using anti-6 × His antibody following His-CaM addition and analyzed by Western blotting with anti-GFP and anti-6 × His antibodies. D , quantitation of the relative intensity of GFP-TSC2 to His-CaM of (C). Graphs represent mean ± SD of three independent experiments.

Article Snippet: For immunofluorescence, TSC2 (D93F12, Cell Signaling Technology #4308) and LAMP2 (H4B4, Proteintech 65053-1-Ig) were used as primary antibodies.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Magnetic Beads, Two Tailed Test, Purification, Western Blot, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Calmodulin enhances mTORC1 signaling by preventing TSC2-Rheb binding

doi: 10.1016/j.jbc.2024.108122

Figure Lengend Snippet: Ca 2+ /CaM alters TSC2-Rheb binding without affecting TSC complex assembly . A , HEK293T cells were mock transfected (m) or transiently transfected with the plasmid encoding Strep-GFP (GFP, −), Strep-GFP-TSC2 (GFP-TSC2) or with both the plasmid encoding HiBiT-Rheb and the plasmid for TSC1-myc. After 24 h of transfection, cells were lysed and respective cell lysates were mixed in the absence (−) or presence of purified wild-type (WT) or Ca 2+ binding-defective mutant (DA) of His-CaM (5.5 μM) together with 100 μM of CaCl 2 . Then, the cell lysates were subjected to immunoprecipitation (IP) using the anti-GFP antibody and analyzed by Western blotting with myc and anti-GFP antibodies. B , quantitation of the relative intensity of GFP-TSC2 to TSC1-myc of (A). Graphs represent mean ± SD of three independent experiments. C , the samples in (A) were also analyzed by HiBiT lytic assay as described in . Graphs represent mean ± SD of three independent experiments. One-way ANOVA with Tukey’s test, ∗∗ p < 0.01.

Article Snippet: For immunofluorescence, TSC2 (D93F12, Cell Signaling Technology #4308) and LAMP2 (H4B4, Proteintech 65053-1-Ig) were used as primary antibodies.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Purification, Mutagenesis, Immunoprecipitation, Western Blot, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Calmodulin enhances mTORC1 signaling by preventing TSC2-Rheb binding

doi: 10.1016/j.jbc.2024.108122

Figure Lengend Snippet: CaM binds the GAP domain of TSC2 in a Ca 2+ -dependent manner in cells . A , schematic representation of CaM-binding peptide substituted R-GECO1 construct. The M13 peptide (CaM binding peptide from myosin light chain) was replaced with the CaM-binding region of TSC2 (residues 1717–1732) or CaM-binding defective mutation ( red ) of TSC2 peptide (WQLQ). B , HeLa cells were transiently transfected with the plasmid encoding R-GECO1(M13 peptide), R-GECO1(TSC2 WT peptide) or R-GECO1(TSC2 (WQLQ) peptide). The medium was replaced with HBSS for 15 min, and then ionomycin (2.5 μM) was added at 105 s. The ratio of fluorescence signals for R-GECO1 to the average signals before ionomycin stimulation (F 0 ) was shown. Graphs are shown as mean ± SD (n = 15). C , HEK293 cells were transferred into HBSS for 15 min, and then ionomycin (2.5 μM) was added. Then, the cells were treated with a crosslinker DSP for 7 min. Cell lysates were subjected to immunoprecipitation with the anti-TSC2 antibody or control IgG. Cell lysates (Input) and immunoprecipitates were analyzed by Western blotting with indicated antibodies. The asterisk indicates IgG heavy chain. D , quantitation of the relative intensity of CaM to TSC2 of (C). Graphs represent mean ± SD of three independent experiments. Two-tailed unpaired Student’s t test, ∗ p < 0.05.

Article Snippet: For immunofluorescence, TSC2 (D93F12, Cell Signaling Technology #4308) and LAMP2 (H4B4, Proteintech 65053-1-Ig) were used as primary antibodies.

Techniques: Binding Assay, Construct, Mutagenesis, Transfection, Plasmid Preparation, Fluorescence, Immunoprecipitation, Control, Western Blot, Quantitation Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Calmodulin enhances mTORC1 signaling by preventing TSC2-Rheb binding

doi: 10.1016/j.jbc.2024.108122

Figure Lengend Snippet: Ca 2+ /CaM affects TSC2 localization without altering TSC2 phosphorylation . A , HEK293 cells were starved of serum (−FBS) for 3 h, pretreated with DMSO or BAPTA-AM (50 μM) or W-7 (30 μM) for the last 1 h (BAPTA-AM) or 5 min (W-7) and then stimulated with or without (−) carbachol (100 μM) for 15 min (+CCh). Cell lysates were analyzed by Western blotting with the indicated antibodies. The images of phospho-T389-S6K1 and S6K1 panels in DMSO and BAPTA-AM lanes are reused from Fig. 6 C . B , Quantitation of the relative intensity of phospho-T1462-TSC2 to total TSC2 of (A), in which the carbachol stimulation (DMSO) was set to 1. Graphs represent mean ± SD of three independent experiments. One-way ANOVA with Tukey’s test. C , HEK293 cells were starved of serum (−FBS) for 3 h, and then treated with carbachol (100 μM) or insulin (100 nM) for 15 min. The cells were then immunostained with anti-TSC2 and anti-LAMP2 antibodies. Merged images of TSC2 ( green ), LAMP2 ( magenta ) and nuclei staining with Hoechst 33342 ( blue ) are also shown. Scale bar, 10 μm. D , colocalizations of TSC2 with LAMP2 were quantified, and Pearson’s correlation coefficient is shown. Data from four independent experiments are shown in blue triangles and were used for statistical analysis. Bars represent averages. The values of Pearson’s correlation coefficient of five individual images in each experiment are also shown in gray circles . One-way ANOVA with Tukey’s test, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: For immunofluorescence, TSC2 (D93F12, Cell Signaling Technology #4308) and LAMP2 (H4B4, Proteintech 65053-1-Ig) were used as primary antibodies.

Techniques: Phospho-proteomics, Western Blot, Quantitation Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: Calmodulin enhances mTORC1 signaling by preventing TSC2-Rheb binding

doi: 10.1016/j.jbc.2024.108122

Figure Lengend Snippet: Overexpression of the CaM-binding region of TSC2 suppresses carbachol-induced mTORC1 activation . A, HEK293 cells were transiently transfected with plasmids expressing GFP (−) or GFP-TSC2 (1717–1732 a.a.) (TSC2 frag) together with the plasmid expressing GST-HA tagged S6K1 (GST-HA-S6K1). After 24 h, cells were starved with serum (−FBS) for 3 h and then stimulated with or without (−) carbachol (100 μM) for 15 min (+CCh). Cell lysates were analyzed by Western blotting with indicated antibodies. B , quantitation of the relative intensity of phospho-T389-S6K1 to total S6K1 of GST-HA-S6K1, in which the condition with carbachol stimulation in GFP expressing cells was set to 1. Graphs represent mean ± SD of three independent experiments. One-way ANOVA with Tukey’s test, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: For immunofluorescence, TSC2 (D93F12, Cell Signaling Technology #4308) and LAMP2 (H4B4, Proteintech 65053-1-Ig) were used as primary antibodies.

Techniques: Over Expression, Binding Assay, Activation Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Calmodulin enhances mTORC1 signaling by preventing TSC2-Rheb binding

doi: 10.1016/j.jbc.2024.108122

Figure Lengend Snippet: A proposed model for Ca 2+ /CaM regulation of mTORC1 via TSC2-Rheb . In the low Ca 2+ condition, most of CaM is not fully occupied by Ca 2+ , and TSC2 can bind and inactivate Rheb by GAP activity of TSC2. In the high Ca 2+ condition, it facilitates CaM binding to TSC2. CaM binding to TSC2 inhibits the interaction between TSC2 and Rheb without affecting TSC complex integrity, and results in the dissociation of TSC2 from the lysosome where Rheb resides. Thus, Ca 2+ /CaM facilitates mTORC1 activation by preventing TSC2 from inactivating Rheb.

Article Snippet: For immunofluorescence, TSC2 (D93F12, Cell Signaling Technology #4308) and LAMP2 (H4B4, Proteintech 65053-1-Ig) were used as primary antibodies.

Techniques: Activity Assay, Binding Assay, Activation Assay

Journal: Circulation research

Article Title: MTORC1-Regulated Metabolism Controlled by TSC2 Limits Cardiac Reperfusion Injury

doi: 10.1161/CIRCRESAHA.120.317710

Figure Lengend Snippet: Major Resources Table

Article Snippet: Nature 2019 https://www.nature.com/articles/s41586-019-0895-y Open in a separate window Antibodies Target antigen Vendor or Source Catalog # Working concentration Lot # (preferred but not required) Persistent ID / URL phospho-p70 S6K (T389) Cell Signaling 9205 1:1,000 26 https://www.cellsignal.com/products/primary-antibodies/phospho-p70-s6-kinase-thr389-antibody/9205 p70 S6K Cell Signaling 9202 1:1,000 20 https://www.cellsignal.com/products/primary-antibodies/p70-s6-kinase-antibody/9202 phospho-4EBP1 (S65) Cell Signaling 9451 1:1,000 14 https://www.cellsignal.com/products/primary-antibodies/phospho-4e-bp1-ser65-antibody/9451 4EBP1 Cell Signaling 9452 1:1,000 12 https://www.cellsignal.com/products/primary-antibodies/4e-bp1-antibody/9452 phospho-ULK1 (S757) (clone D706U) Cell Signaling 14202 1:500 4 https://www.cellsignal.com/products/primary-antibodies/phospho-ulk1-ser757-d7o6u-rabbit-mab/14202?site-search-type=Products ULK1 (clone D8H5) Cell Signaling 8054 1:500 5 https://www.cellsignal.com/products/primary-antibodies/ulk1-d8h5-rabbit-mab/8054 TSC2 (clone D93F12) Cell Signaling 3612 1:1,000 6 https://www.cellsignal.com/products/primary-antibodies/tuberin-tsc2-antibody/3612 phospho-TSC2 (S1365) NovoPro Labs 120718 1:500 NFSA120720AH https://www.novoprolabs.com/p/phospho-mouse-tsc2-s1365-antibody-120718.html Total Protein Stain Li-Cor 926–11016 5–10ml per membrane C80522 –02 https://www.licor.com/bio/reagents/revert-700-total-protein-stain-kits Open in a separate window DNA/cDNA Clones Clone Name Sequence Source / Repository Persistent ID / URL Ad - TSC2 WT or TSC2 S1365E or TSC2 S1365A N-terminally HA-tagged human TSC2 WT, S1365E, S1365A adenovirus, and N-terminally HA-tagged wild-type sequence or human TSC2(S1365A) or TSC2(S1365E) adenovirus plasmid encoding human HA-tagged TSC2 WT or TSC2 S1365E or TSC2 S1365A (Ranek et al.

Techniques:

Journal: Circulation research

Article Title: MTORC1-Regulated Metabolism Controlled by TSC2 Limits Cardiac Reperfusion Injury

doi: 10.1161/CIRCRESAHA.120.317710

Figure Lengend Snippet: (A) Immunoblot and summary data for total and pS1365 TSC2 for sham, IS, IR, (n=6/group) and IR+IPC (n=5). KW test (p=0.01), DMTC (2-comparison test): * p=0.07; ** 0.003. (B) Percent change in isolated heart rate-pressure product (RPP) after IR in TSC2WT +/− IPC, and TSC2SE + IPC. (n/group in figure; one outlier excluded in SE+IPC by ROUT outlier test (Q=1%). KW (p=1e−5, DMCT (3-way comparison): *p=0.017, ** 5e−6. (C) Example immunoblots and (D) summary data for mTORC1 activation substrates 4EBP1, S6K, and Ulk1 from TSC2WT and TSC2SE ex vivo hearts pre-treated with IPC and then subjected to IR. Summary results for n=4/group, WT vs SE compared by Mann-Whitney test * p=0.028). The dark and light gray shaded areas show 90% confidence bands for comparison results for WT+IR and SE+IR respectively - without IPC, as derived from data provided in Figure 1B. Each data symbol within groups represents a biological replicate.

Article Snippet: Nature 2019 https://www.nature.com/articles/s41586-019-0895-y Open in a separate window Antibodies Target antigen Vendor or Source Catalog # Working concentration Lot # (preferred but not required) Persistent ID / URL phospho-p70 S6K (T389) Cell Signaling 9205 1:1,000 26 https://www.cellsignal.com/products/primary-antibodies/phospho-p70-s6-kinase-thr389-antibody/9205 p70 S6K Cell Signaling 9202 1:1,000 20 https://www.cellsignal.com/products/primary-antibodies/p70-s6-kinase-antibody/9202 phospho-4EBP1 (S65) Cell Signaling 9451 1:1,000 14 https://www.cellsignal.com/products/primary-antibodies/phospho-4e-bp1-ser65-antibody/9451 4EBP1 Cell Signaling 9452 1:1,000 12 https://www.cellsignal.com/products/primary-antibodies/4e-bp1-antibody/9452 phospho-ULK1 (S757) (clone D706U) Cell Signaling 14202 1:500 4 https://www.cellsignal.com/products/primary-antibodies/phospho-ulk1-ser757-d7o6u-rabbit-mab/14202?site-search-type=Products ULK1 (clone D8H5) Cell Signaling 8054 1:500 5 https://www.cellsignal.com/products/primary-antibodies/ulk1-d8h5-rabbit-mab/8054 TSC2 (clone D93F12) Cell Signaling 3612 1:1,000 6 https://www.cellsignal.com/products/primary-antibodies/tuberin-tsc2-antibody/3612 phospho-TSC2 (S1365) NovoPro Labs 120718 1:500 NFSA120720AH https://www.novoprolabs.com/p/phospho-mouse-tsc2-s1365-antibody-120718.html Total Protein Stain Li-Cor 926–11016 5–10ml per membrane C80522 –02 https://www.licor.com/bio/reagents/revert-700-total-protein-stain-kits Open in a separate window DNA/cDNA Clones Clone Name Sequence Source / Repository Persistent ID / URL Ad - TSC2 WT or TSC2 S1365E or TSC2 S1365A N-terminally HA-tagged human TSC2 WT, S1365E, S1365A adenovirus, and N-terminally HA-tagged wild-type sequence or human TSC2(S1365A) or TSC2(S1365E) adenovirus plasmid encoding human HA-tagged TSC2 WT or TSC2 S1365E or TSC2 S1365A (Ranek et al.

Techniques: Western Blot, Comparison, Isolation, Activation Assay, Ex Vivo, MANN-WHITNEY, Derivative Assay

Journal: Circulation research

Article Title: MTORC1-Regulated Metabolism Controlled by TSC2 Limits Cardiac Reperfusion Injury

doi: 10.1161/CIRCRESAHA.120.317710

Figure Lengend Snippet: (A) Baseline mitochondrial respiration in isolated neonatal rat ventricular myocytes (NRVMs) infected with adenovirus expressing either TSC2 WT, SA, or SE (n=30, 29, 27, respectively, KW test, DMCT: p=values in panels). (B) Mean time-tracings for stress-test protocol (see methods) comparing each group. (C) Mitochondrial respiration after ischemia/reoxygenation (n=30, 31, 28/group,respectively), KW test, DMCT, p-values in panels. (D) Myocardial long-chain acyl-carnitines from isolated hearts during ischemia (n = 5, 4, 6 for WT, SE, SA, respectively , KW test (p-values in upper left of each panel), DMCT (WT or SA vs SE: p values shown). Biological replicates indicated in figure. ATP: adenosine triphosphate, OCR: oxygen consumption rate, ECAR: extracellular acidification rate. Each data dot symbol within groups reflects a biological replicate.

Article Snippet: Nature 2019 https://www.nature.com/articles/s41586-019-0895-y Open in a separate window Antibodies Target antigen Vendor or Source Catalog # Working concentration Lot # (preferred but not required) Persistent ID / URL phospho-p70 S6K (T389) Cell Signaling 9205 1:1,000 26 https://www.cellsignal.com/products/primary-antibodies/phospho-p70-s6-kinase-thr389-antibody/9205 p70 S6K Cell Signaling 9202 1:1,000 20 https://www.cellsignal.com/products/primary-antibodies/p70-s6-kinase-antibody/9202 phospho-4EBP1 (S65) Cell Signaling 9451 1:1,000 14 https://www.cellsignal.com/products/primary-antibodies/phospho-4e-bp1-ser65-antibody/9451 4EBP1 Cell Signaling 9452 1:1,000 12 https://www.cellsignal.com/products/primary-antibodies/4e-bp1-antibody/9452 phospho-ULK1 (S757) (clone D706U) Cell Signaling 14202 1:500 4 https://www.cellsignal.com/products/primary-antibodies/phospho-ulk1-ser757-d7o6u-rabbit-mab/14202?site-search-type=Products ULK1 (clone D8H5) Cell Signaling 8054 1:500 5 https://www.cellsignal.com/products/primary-antibodies/ulk1-d8h5-rabbit-mab/8054 TSC2 (clone D93F12) Cell Signaling 3612 1:1,000 6 https://www.cellsignal.com/products/primary-antibodies/tuberin-tsc2-antibody/3612 phospho-TSC2 (S1365) NovoPro Labs 120718 1:500 NFSA120720AH https://www.novoprolabs.com/p/phospho-mouse-tsc2-s1365-antibody-120718.html Total Protein Stain Li-Cor 926–11016 5–10ml per membrane C80522 –02 https://www.licor.com/bio/reagents/revert-700-total-protein-stain-kits Open in a separate window DNA/cDNA Clones Clone Name Sequence Source / Repository Persistent ID / URL Ad - TSC2 WT or TSC2 S1365E or TSC2 S1365A N-terminally HA-tagged human TSC2 WT, S1365E, S1365A adenovirus, and N-terminally HA-tagged wild-type sequence or human TSC2(S1365A) or TSC2(S1365E) adenovirus plasmid encoding human HA-tagged TSC2 WT or TSC2 S1365E or TSC2 S1365A (Ranek et al.

Techniques: Isolation, Infection, Expressing

Journal: Circulation research

Article Title: MTORC1-Regulated Metabolism Controlled by TSC2 Limits Cardiac Reperfusion Injury

doi: 10.1161/CIRCRESAHA.120.317710

Figure Lengend Snippet: Major Resources Table

Article Snippet: Nature 2019 https://www.nature.com/articles/s41586-019-0895-y Open in a separate window Antibodies Target antigen Vendor or Source Catalog # Working concentration Lot # (preferred but not required) Persistent ID / URL phospho-p70 S6K (T389) Cell Signaling 9205 1:1,000 26 https://www.cellsignal.com/products/primary-antibodies/phospho-p70-s6-kinase-thr389-antibody/9205 p70 S6K Cell Signaling 9202 1:1,000 20 https://www.cellsignal.com/products/primary-antibodies/p70-s6-kinase-antibody/9202 phospho-4EBP1 (S65) Cell Signaling 9451 1:1,000 14 https://www.cellsignal.com/products/primary-antibodies/phospho-4e-bp1-ser65-antibody/9451 4EBP1 Cell Signaling 9452 1:1,000 12 https://www.cellsignal.com/products/primary-antibodies/4e-bp1-antibody/9452 phospho-ULK1 (S757) (clone D706U) Cell Signaling 14202 1:500 4 https://www.cellsignal.com/products/primary-antibodies/phospho-ulk1-ser757-d7o6u-rabbit-mab/14202?site-search-type=Products ULK1 (clone D8H5) Cell Signaling 8054 1:500 5 https://www.cellsignal.com/products/primary-antibodies/ulk1-d8h5-rabbit-mab/8054 TSC2 (clone D93F12) Cell Signaling 3612 1:1,000 6 https://www.cellsignal.com/products/primary-antibodies/tuberin-tsc2-antibody/3612 phospho-TSC2 (S1365) NovoPro Labs 120718 1:500 NFSA120720AH https://www.novoprolabs.com/p/phospho-mouse-tsc2-s1365-antibody-120718.html Total Protein Stain Li-Cor 926–11016 5–10ml per membrane C80522 –02 https://www.licor.com/bio/reagents/revert-700-total-protein-stain-kits Open in a separate window DNA/cDNA Clones Clone Name Sequence Source / Repository Persistent ID / URL Ad - TSC2 WT or TSC2 S1365E or TSC2 S1365A N-terminally HA-tagged human TSC2 WT, S1365E, S1365A adenovirus, and N-terminally HA-tagged wild-type sequence or human TSC2(S1365A) or TSC2(S1365E) adenovirus plasmid encoding human HA-tagged TSC2 WT or TSC2 S1365E or TSC2 S1365A (Ranek et al.

Techniques: