Journal: Nature Communications

Article Title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

doi: 10.1038/s41467-023-36881-7

Figure Lengend Snippet: Enhanced Cd276 promoter activity in Tsc2−/− 105K cells expressing empty vector (EV) compared to reconstitution of TSC2 ( a ) and in Tsc2 KO MEFs compared to Tsc2 -WT MEFs ( b ). Relative luciferase activity was determined by a dual-luciferase assay system. psiCHECK2-Cd276 encodes the Cd276 promoter. Empty psiCHECK2 vector was used as the negative control. n = 6, means ± SD, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, *** p < 0.001. Raptor, mTOR or S6K knockdown suppresses Cd276 promoter activity in Tsc2−/− 105K cells ( c ) and Tsc2 KO MEFs ( d ). n = 6, means ± SD, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, ** p < 0.01, **** p < 0.0001. siRNA knockdown of YY2 reduces B7-H3 protein expression in Tsc2−/− 105K cells ( e ) and Tsc2 KO MEFs ( f ) ( n = 3). Knockdown of YY2 reduces Cd276 mRNA expression in Tsc2−/− 105K cells ( g ) and Tsc2 KO MEFs ( h ). n = 3, means ± SD, two-tailed unpaired Student’s t -test, * p < 0.05, *** p < 0.001. i Promoter region of Cd276 displaying the location of the YY2 binding site. YY2 ChIP-qPCR analysis showing increased YY2 occupancy on the Cd276 promoter in Tsc2−/− 105K cells ( j ) and Tsc2 KO MEFs ( k ). n = 4, means ± SEM, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Cd276 promoter activity is suppressed by YY2 knockdown in Tsc2−/− 105K cells ( l ) and Tsc2 KO MEFs ( m ). n = 6, means ± SD, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, *** p < 0.001. Source data and exact p values are provided in the Source data file.

Article Snippet: Plasmids encoding wild-type YY2 (Myc-DDK-YY2, Cat#MR218884) was purchased from Origene, USA, wild-type S6K (pRK7-HA-S6K1-WT, Cat# 8984), kinase-dead S6K (pRK7-HA-S6K1-KR, Cat# 8985), wild-type Smurf1 (pCMV5B-Flag-Smurf1 wt, Cat# 11752), wild-type mTOR (pcDNA3-FLAG-MTOR, Cat#26603), L1460P mutant mTOR (pcDNA3-FLAG-MTOR-L1460P, Cat#69006), C1483F mutant mTOR (pcDNA3-FLAG-MTOR-C1483F, Cat#69008), S2215Y mutant mTOR (pcDNA3-FLAG-MTOR-S2215Y, Cat#69013), and R2505P mutant mTOR (pcDNA3-FLAG-MTOR-R2505P, Cat#69015) were purchased from Addgene, USA.

Techniques: Activity Assay, Expressing, Plasmid Preparation, Luciferase, Negative Control, Knockdown, Two Tailed Test, Binding Assay, ChIP-qPCR

Journal: Nature Communications

Article Title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

doi: 10.1038/s41467-023-36881-7

Figure Lengend Snippet: YY2 protein expression in Tsc2−/− 105K cells ( a ) and Tsc2 KO MEFs ( b ) treated with 20 nM rapamycin (Rapa) or vehicle for 24 hr ( n = 3). YY2 protein expression in Tsc2−/− 105K cells ( c ) and Tsc2 KO MEFs ( d ) treated with 10 μM PF-4708671 (S6K1 inhibitor) or vehicle for 24 hr ( n = 3). e Immunoblot analysis of HeLa cells transfected with eGFP-YY2, wild-type S6K (HA-WT-S6K), or kinase-dead S6K (HA-KD-S6K) for 48 hr ( n = 3). f Immunoblot analysis of in vitro kinase assays of recombinant active S6K in the presence or absence of myc-YY2 as a substrate. eIF4B was used as a positive control ( n = 3). g Immunoblot analysis of immunoprecipitated (IP) GFP-YY2 and the whole-cell-lysate (WCL) from HeLa cells expressing GFP-YY2 ( n = 3). h Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells expressing GFP-YY2 ( n = 3). i The evolutionarily conserved putative S6K phosphorylation site in YY2. j Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells stably expressing wild-type (WT) GFP-YY2 or mutant (T336A) GFP-YY2, treated with 40 μM PF-4708671 for 1 hr ( n = 3). k Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells expressing WT GFP-YY2 or T336A GFP-YY2 ( n = 3). l , m Network view of predicted E3 ubiquitin ligase-YY2 interactions by UbiBrowser 2.0. Confidence level of ( m ). n Decreased EGFP-YY2 expression in HeLa cells transfected with Smurf1-FLAG ( n = 3). o Decreased Smurf1 ubiquitination in HeLa cells transfected with S6K. Immunoblot analysis of GFP-immunoprecipitates from EGFP-YY2 HeLa cells transfected with Smurf1-FLAG or HA-WT-S6K for 24 hr ( n = 3). p Immunoblot analysis of GFP-and IgG control immunoprecipitates from EGFP-YY2 HeLa cells transfected with Smurf1-FLAG ( n = 3). q Immunoblot analysis of B7-H3 expression in Tsc2−/− 105K cells, transfected with control or myc-YY2 for 48 hr and treated with 20 nM rapamycin or vehicle for the last 24 hr ( n = 3). Source data is provided in the Source data file.

Article Snippet: Plasmids encoding wild-type YY2 (Myc-DDK-YY2, Cat#MR218884) was purchased from Origene, USA, wild-type S6K (pRK7-HA-S6K1-WT, Cat# 8984), kinase-dead S6K (pRK7-HA-S6K1-KR, Cat# 8985), wild-type Smurf1 (pCMV5B-Flag-Smurf1 wt, Cat# 11752), wild-type mTOR (pcDNA3-FLAG-MTOR, Cat#26603), L1460P mutant mTOR (pcDNA3-FLAG-MTOR-L1460P, Cat#69006), C1483F mutant mTOR (pcDNA3-FLAG-MTOR-C1483F, Cat#69008), S2215Y mutant mTOR (pcDNA3-FLAG-MTOR-S2215Y, Cat#69013), and R2505P mutant mTOR (pcDNA3-FLAG-MTOR-R2505P, Cat#69015) were purchased from Addgene, USA.

Techniques: Expressing, Western Blot, Transfection, In Vitro, Recombinant, Positive Control, Immunoprecipitation, Phospho-proteomics, Stable Transfection, Mutagenesis, Ubiquitin Proteomics, Control

Journal: Nature Communications

Article Title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

doi: 10.1038/s41467-023-36881-7

Figure Lengend Snippet: YY2 protein expression in Tsc2−/− 105K cells ( a ) and Tsc2 KO MEFs ( b ) treated with 20 nM rapamycin (Rapa) or vehicle for 24 hr ( n = 3). YY2 protein expression in Tsc2−/− 105K cells ( c ) and Tsc2 KO MEFs ( d ) treated with 10 μM PF-4708671 (S6K1 inhibitor) or vehicle for 24 hr ( n = 3). e Immunoblot analysis of HeLa cells transfected with eGFP-YY2, wild-type S6K (HA-WT-S6K), or kinase-dead S6K (HA-KD-S6K) for 48 hr ( n = 3). f Immunoblot analysis of in vitro kinase assays of recombinant active S6K in the presence or absence of myc-YY2 as a substrate. eIF4B was used as a positive control ( n = 3). g Immunoblot analysis of immunoprecipitated (IP) GFP-YY2 and the whole-cell-lysate (WCL) from HeLa cells expressing GFP-YY2 ( n = 3). h Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells expressing GFP-YY2 ( n = 3). i The evolutionarily conserved putative S6K phosphorylation site in YY2. j Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells stably expressing wild-type (WT) GFP-YY2 or mutant (T336A) GFP-YY2, treated with 40 μM PF-4708671 for 1 hr ( n = 3). k Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells expressing WT GFP-YY2 or T336A GFP-YY2 ( n = 3). l , m Network view of predicted E3 ubiquitin ligase-YY2 interactions by UbiBrowser 2.0. Confidence level of ( m ). n Decreased EGFP-YY2 expression in HeLa cells transfected with Smurf1-FLAG ( n = 3). o Decreased Smurf1 ubiquitination in HeLa cells transfected with S6K. Immunoblot analysis of GFP-immunoprecipitates from EGFP-YY2 HeLa cells transfected with Smurf1-FLAG or HA-WT-S6K for 24 hr ( n = 3). p Immunoblot analysis of GFP-and IgG control immunoprecipitates from EGFP-YY2 HeLa cells transfected with Smurf1-FLAG ( n = 3). q Immunoblot analysis of B7-H3 expression in Tsc2−/− 105K cells, transfected with control or myc-YY2 for 48 hr and treated with 20 nM rapamycin or vehicle for the last 24 hr ( n = 3). Source data is provided in the Source data file.

Article Snippet: Plasmids encoding wild-type YY2 (Myc-DDK-YY2, Cat#MR218884) was purchased from Origene, USA, wild-type S6K (pRK7-HA-S6K1-WT, Cat# 8984), kinase-dead S6K (pRK7-HA-S6K1-KR, Cat# 8985), wild-type Smurf1 (pCMV5B-Flag-Smurf1 wt, Cat# 11752), wild-type mTOR (pcDNA3-FLAG-MTOR, Cat#26603), L1460P mutant mTOR (pcDNA3-FLAG-MTOR-L1460P, Cat#69006), C1483F mutant mTOR (pcDNA3-FLAG-MTOR-C1483F, Cat#69008), S2215Y mutant mTOR (pcDNA3-FLAG-MTOR-S2215Y, Cat#69013), and R2505P mutant mTOR (pcDNA3-FLAG-MTOR-R2505P, Cat#69015) were purchased from Addgene, USA.

Techniques: Expressing, Western Blot, Transfection, In Vitro, Recombinant, Positive Control, Immunoprecipitation, Phospho-proteomics, Stable Transfection, Mutagenesis, Ubiquitin Proteomics, Control

Journal: Nature Communications

Article Title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

doi: 10.1038/s41467-023-36881-7

Figure Lengend Snippet: Kaplan-Meier plot of overall patient survival stratified by high ( n = 2076) and low ( n = 2185) CD276 expression ( a ), high ( n = 2001) and low ( n = 2268) mTORC1 score ( b ), and high CD276 expression plus high mTORC1 score ( n = 1296) and low CD276 expression plus low mTORC1 score ( n = 1160) ( c ). Log-rank test. Forest plot of hazard ratios by tumor type and overall (with 95% confidence intervals) stratified by high and low CD276 expression ( d ), high and low mTORC1 score ( e ), and high and low CD276 expression plus high and low mTORC1 score ( f ). Hazard ratio greater than 1 (red) indicates an association with worse outcome. COX proportional hazards regression with Efron’s approximation. g , h Representative images of strong and weak immunohistochemical staining of phospho-S6 (Ser240/244) (pS6) and B7-H3 in tumor tissue microarrays, which include 12 cancer types: meningothelial meningioma, breast infiltrating ductal adenocarcinoma, colon adenocarcinoma, stomach adenocarcinoma, prostate adenocarcinoma, uterus adenocarcinoma, ovarian adenocarcinoma, esophageal squamous cell carcinomas, kidney clear cell carcinomas, hepatocellular carcinomas, lung adenocarcinoma/squamous cell carcinomas, and bladder transitional/papillary cell carcinomas. Scale bar = 50 μm ( g ). Correlation analysis between the phospho-S6 immunoreactivity score and B7-H3 expression score in the tumors in g (Pearson correlation coefficient test) ( h ). i , j Representative images of strong and weak immunohistochemical staining of phospho-S6K (Thr389) (pS6K) and B7-H3 in tumor tissue microarrays, which include 12 cancer types indicated in ( g ). Scale bar = 50 μm ( i ). Correlation analysis between the phospho-S6K immunoreactivity score and B7-H3 expression score in the tumors in i (Pearson correlation coefficient test) ( j ). mTORC1 score ( k ) and CD276 expression (l ) among the six immune subtypes. Bars indicate median line, boxes denote interquartile range, whiskers denote 1–99 percentile and outliers are shown. Nonparametric Kruskal-Wallis test with Dunn’s multiple comparisons test. n = 5429, **** p < 0.0001. m Heatmap indicates mean scores of immune-cell of all patients in high CD276 and high mTORC1 tumors (left) and low CD276 - low mTORC1 tumors (right). Source data is provided in the Source data file.

Article Snippet: Plasmids encoding wild-type YY2 (Myc-DDK-YY2, Cat#MR218884) was purchased from Origene, USA, wild-type S6K (pRK7-HA-S6K1-WT, Cat# 8984), kinase-dead S6K (pRK7-HA-S6K1-KR, Cat# 8985), wild-type Smurf1 (pCMV5B-Flag-Smurf1 wt, Cat# 11752), wild-type mTOR (pcDNA3-FLAG-MTOR, Cat#26603), L1460P mutant mTOR (pcDNA3-FLAG-MTOR-L1460P, Cat#69006), C1483F mutant mTOR (pcDNA3-FLAG-MTOR-C1483F, Cat#69008), S2215Y mutant mTOR (pcDNA3-FLAG-MTOR-S2215Y, Cat#69013), and R2505P mutant mTOR (pcDNA3-FLAG-MTOR-R2505P, Cat#69015) were purchased from Addgene, USA.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Nature Communications

Article Title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

doi: 10.1038/s41467-023-36881-7

Figure Lengend Snippet: B7-H3 protein expression in Tsc2−/− 105K cells with stable reconstitution of TSC2 or empty vector (EV) ( a ), TSC2−/− 621-101 angiomyolipoma-derived cells with reconstitution of TSC2 or empty vector (EV) ( b ), and Tsc2 -WT and Tsc2 KO MEFs ( c ). For all figure legends, n = 3 indicates representative of 3 biologic samples ( n = 3). B7-H3 mRNA expression in Tsc2−/− 105K cells with stable reconstitution of TSC2 or empty vector (EV) ( d ), TSC2−/− 621-101 angiomyolipoma tumor cells with reconstitution of TSC2 or empty vector (EV) ( e ), Tsc2 -WT and Tsc2 KO MEFs ( f ). Means ± SD, two-tailed unpaired Student’s t -test ( d , f ) or one-way ANOVA with Dunnett’s multiple comparisons test ( e ), * p < 0.05, ** p < 0.01. n = 3. B7-H3 protein expression in Tsc2−/− 105K cells ( g ), TSC2−/− 621-101 angiomyolipoma tumor cells ( h ), and Tsc2 KO MEFs ( i ) treated with 20 nM rapamycin (Rapa), 500 nM Torin 1 or vehicle for 24 hr ( n = 3). B7-H3 mRNA expression in Tsc2−/− 105K cells ( j ), TSC2−/− 621-101 angiomyolipoma tumor cells ( k ), and Tsc2 KO MEFs ( l ) treated with 20 nM rapamycin (Rapa), 500 nM Torin 1, or vehicle for 24 hr. n = 3, means ± SD, one-way ANOVA with Dunnett’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001. m Immunoblotting analysis of Tsc2 -WT and Tsc2 KO MEFs transfected with non-targeting control siRNA (Ctrl) or SMARTpool siRNAs targeting Raptor, Rictor, mTOR, S6K, or 4E-BP1 for 48 h hr ( n = 3). n qRT-PCR analysis of Tsc2 -WT and Tsc2 KO MEFs transfected with non-targeting control siRNA (Ctrl) or SMARTpool siRNAs targeting either Raptor, Rictor, mTOR, S6K, or 4E-BP1 for 48 hr. n = 3, means ± SD, two-way ANOVA with Dunnett’s multiple comparisons test, *** p < 0.001, **** p < 0.0001. Source data and exact p values are provided in the Source data file.

Article Snippet: Plasmids encoding wild-type YY2 (Myc-DDK-YY2, Cat#MR218884) was purchased from Origene, USA, wild-type S6K (pRK7-HA-S6K1-WT, Cat# 8984), kinase-dead S6K (pRK7-HA-S6K1-KR, Cat# 8985), wild-type Smurf1 (pCMV5B-Flag-Smurf1 wt, Cat# 11752), wild-type mTOR (pcDNA3-FLAG-MTOR, Cat#26603), L1460P mutant mTOR (pcDNA3-FLAG-MTOR-L1460P, Cat#69006), C1483F mutant mTOR (pcDNA3-FLAG-MTOR-C1483F, Cat#69008), S2215Y mutant mTOR (pcDNA3-FLAG-MTOR-S2215Y, Cat#69013), and R2505P mutant mTOR (pcDNA3-FLAG-MTOR-R2505P, Cat#69015) were purchased from Addgene, USA.

Techniques: Expressing, Plasmid Preparation, Derivative Assay, Two Tailed Test, Western Blot, Transfection, Control, Quantitative RT-PCR

Journal: Nature Communications

Article Title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

doi: 10.1038/s41467-023-36881-7

Figure Lengend Snippet: Enhanced Cd276 promoter activity in Tsc2−/− 105K cells expressing empty vector (EV) compared to reconstitution of TSC2 ( a ) and in Tsc2 KO MEFs compared to Tsc2 -WT MEFs ( b ). Relative luciferase activity was determined by a dual-luciferase assay system. psiCHECK2-Cd276 encodes the Cd276 promoter. Empty psiCHECK2 vector was used as the negative control. n = 6, means ± SD, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, *** p < 0.001. Raptor, mTOR or S6K knockdown suppresses Cd276 promoter activity in Tsc2−/− 105K cells ( c ) and Tsc2 KO MEFs ( d ). n = 6, means ± SD, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, ** p < 0.01, **** p < 0.0001. siRNA knockdown of YY2 reduces B7-H3 protein expression in Tsc2−/− 105K cells ( e ) and Tsc2 KO MEFs ( f ) ( n = 3). Knockdown of YY2 reduces Cd276 mRNA expression in Tsc2−/− 105K cells ( g ) and Tsc2 KO MEFs ( h ). n = 3, means ± SD, two-tailed unpaired Student’s t -test, * p < 0.05, *** p < 0.001. i Promoter region of Cd276 displaying the location of the YY2 binding site. YY2 ChIP-qPCR analysis showing increased YY2 occupancy on the Cd276 promoter in Tsc2−/− 105K cells ( j ) and Tsc2 KO MEFs ( k ). n = 4, means ± SEM, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Cd276 promoter activity is suppressed by YY2 knockdown in Tsc2−/− 105K cells ( l ) and Tsc2 KO MEFs ( m ). n = 6, means ± SD, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, *** p < 0.001. Source data and exact p values are provided in the Source data file.

Article Snippet: Plasmids encoding wild-type YY2 (Myc-DDK-YY2, Cat#MR218884) was purchased from Origene, USA, wild-type S6K (pRK7-HA-S6K1-WT, Cat# 8984), kinase-dead S6K (pRK7-HA-S6K1-KR, Cat# 8985), wild-type Smurf1 (pCMV5B-Flag-Smurf1 wt, Cat# 11752), wild-type mTOR (pcDNA3-FLAG-MTOR, Cat#26603), L1460P mutant mTOR (pcDNA3-FLAG-MTOR-L1460P, Cat#69006), C1483F mutant mTOR (pcDNA3-FLAG-MTOR-C1483F, Cat#69008), S2215Y mutant mTOR (pcDNA3-FLAG-MTOR-S2215Y, Cat#69013), and R2505P mutant mTOR (pcDNA3-FLAG-MTOR-R2505P, Cat#69015) were purchased from Addgene, USA.

Techniques: Activity Assay, Expressing, Plasmid Preparation, Luciferase, Negative Control, Knockdown, Two Tailed Test, Binding Assay, ChIP-qPCR

Journal: Nature Communications

Article Title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

doi: 10.1038/s41467-023-36881-7

Figure Lengend Snippet: YY2 protein expression in Tsc2−/− 105K cells ( a ) and Tsc2 KO MEFs ( b ) treated with 20 nM rapamycin (Rapa) or vehicle for 24 hr ( n = 3). YY2 protein expression in Tsc2−/− 105K cells ( c ) and Tsc2 KO MEFs ( d ) treated with 10 μM PF-4708671 (S6K1 inhibitor) or vehicle for 24 hr ( n = 3). e Immunoblot analysis of HeLa cells transfected with eGFP-YY2, wild-type S6K (HA-WT-S6K), or kinase-dead S6K (HA-KD-S6K) for 48 hr ( n = 3). f Immunoblot analysis of in vitro kinase assays of recombinant active S6K in the presence or absence of myc-YY2 as a substrate. eIF4B was used as a positive control ( n = 3). g Immunoblot analysis of immunoprecipitated (IP) GFP-YY2 and the whole-cell-lysate (WCL) from HeLa cells expressing GFP-YY2 ( n = 3). h Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells expressing GFP-YY2 ( n = 3). i The evolutionarily conserved putative S6K phosphorylation site in YY2. j Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells stably expressing wild-type (WT) GFP-YY2 or mutant (T336A) GFP-YY2, treated with 40 μM PF-4708671 for 1 hr ( n = 3). k Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells expressing WT GFP-YY2 or T336A GFP-YY2 ( n = 3). l , m Network view of predicted E3 ubiquitin ligase-YY2 interactions by UbiBrowser 2.0. Confidence level of ( m ). n Decreased EGFP-YY2 expression in HeLa cells transfected with Smurf1-FLAG ( n = 3). o Decreased Smurf1 ubiquitination in HeLa cells transfected with S6K. Immunoblot analysis of GFP-immunoprecipitates from EGFP-YY2 HeLa cells transfected with Smurf1-FLAG or HA-WT-S6K for 24 hr ( n = 3). p Immunoblot analysis of GFP-and IgG control immunoprecipitates from EGFP-YY2 HeLa cells transfected with Smurf1-FLAG ( n = 3). q Immunoblot analysis of B7-H3 expression in Tsc2−/− 105K cells, transfected with control or myc-YY2 for 48 hr and treated with 20 nM rapamycin or vehicle for the last 24 hr ( n = 3). Source data is provided in the Source data file.

Article Snippet: Plasmids encoding wild-type YY2 (Myc-DDK-YY2, Cat#MR218884) was purchased from Origene, USA, wild-type S6K (pRK7-HA-S6K1-WT, Cat# 8984), kinase-dead S6K (pRK7-HA-S6K1-KR, Cat# 8985), wild-type Smurf1 (pCMV5B-Flag-Smurf1 wt, Cat# 11752), wild-type mTOR (pcDNA3-FLAG-MTOR, Cat#26603), L1460P mutant mTOR (pcDNA3-FLAG-MTOR-L1460P, Cat#69006), C1483F mutant mTOR (pcDNA3-FLAG-MTOR-C1483F, Cat#69008), S2215Y mutant mTOR (pcDNA3-FLAG-MTOR-S2215Y, Cat#69013), and R2505P mutant mTOR (pcDNA3-FLAG-MTOR-R2505P, Cat#69015) were purchased from Addgene, USA.

Techniques: Expressing, Western Blot, Transfection, In Vitro, Recombinant, Positive Control, Immunoprecipitation, Phospho-proteomics, Stable Transfection, Mutagenesis, Ubiquitin Proteomics, Control