p tuberin tsc2  (Cell Signaling Technology Inc)

Journal: EMBO Molecular Medicine

Article Title: The critical role of the proto-oncogene c- Kit in TSC renal cystogenesis

doi: 10.1038/s44321-025-00360-x

Figure Lengend Snippet: The timeline of c-KIT expression and cystogenesis in the kidneys of Tsc2 +/− mice was examined. ( A ) H&E images (20×) of the kidneys of WT and Tsc2 +/− mice taken 6, 10, and 15 months of age. ( B ) Northern blot images and corresponding Image J analysis results comparing the expression of c-Kit (top panel) and Foxi1 (middle panel) in the kidneys of Tsc2 +/− mice at 6, 10, and 15 months of age ( n = 2/group/timepoint). Results are presented as average ± SD. ( C ) Representative immunofluorescence microscopic images showing the co-localization of c-KIT (red; left panel) and H + -ATPase (green; right panel) in the kidneys of WT and Tsc2 +/− mice. The middle panel depicts a merged image of c-KIT and H + -ATPase localization. White arrow points to basolateral c-KIT expression and the yellow arrow indicates the apical localization H + -ATPase. “C” represents a cyst. Scale bar equals 50 μm ( B ) and 20 μm ( C ). .

Article Snippet: The following antibodies were used for Western blot experiments: (1) tyr719 p-c-KIT (1:1000) (Cell Signaling Technology, Danvers, MA); (2) ser664 p-TSC2 (1:1000) (Invitrogen, Rockford, IL); (3) ser939 p-TSC2 (1:1000) (Cell Signaling Technology, Danvers, MA); (4) β-actin (1:1000) (Santa Cruz Biotechnology, Dallas, TX); (5) TSC1 (1:2000) (Protein Tech, Rosemont, IL); (6) TSC2 (1:2000) (Protein Tech, Rosemont, IL); (7) ERK1/2--WB (1:1000) (Cell Signaling Technology, Danvers, MA); (8) RSK1 WB (1:1000) (Cell Signaling Technology, Danvers, MA); (9) AKT WB (1:1000) (Cell Signaling Technology, Danvers, MA); and (10) S6 ribosomal (1:1000) (Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Northern Blot, Immunofluorescence

Journal: EMBO Molecular Medicine

Article Title: The critical role of the proto-oncogene c- Kit in TSC renal cystogenesis

doi: 10.1038/s44321-025-00360-x

Figure Lengend Snippet: RAS-MAPK signaling and TSC2 phosphorylation/inactivation were examined in the kidneys of WT and Tsc1 - KO mice. ( A ) Phosphorylation of ERK1/2, RSK1, and AKT were compared in WT and 28- and 45-day-old Tsc1 - KO mice ( n = 6/group). Image J analysis of the results (histogram) indicates that the phosphorylation of all three kinases is significantly elevated in day 28 kidneys of Tsc1-KO compared to WT mice (pERK1/2/ERK1/2, p = 0.009406302; pAKT/AKT, p = 0.007124116; and pRSK1/RSK1, p = 0.0003883); while in day 45 samples only the phosphorylation of ERK1/2 and RSK1 are significantly higher in the kidneys of Tsc1 - KO than that of WT mice (pERK1/2/ERK1/2, p = 0.016311363; and pRSK1/RSK1, p = 0.00644616). Results are presented as average ± SD. ( B ) Immunohistochemical labeling of p-ERK1/2, p-RSK1, and p-AKT in 45-day-old WT and Tsc1-KO mice. An increase in labeling can be seen within the cyst epithelium for all three phosphorylated kinases. “C” represents cysts. Scale bar equals 20 μm. ( C ) Phosphorylation of TSC2 by ERK1/2, AKT and RSK1 leads to its inactivation and allows unregulated mTORC1 driven cell proliferation (see the results in Fig. ). Phosphorylation of TSC2 on Serine residues 664, 939, and 1798 was compared in WT and Tsc1 - KO mice. Image J analysis of the results (histogram) indicates that the phosphorylation raito of 664Ser p-TSC2/TSC2 is significantly ( p = 1.78005 × 10 −5 ) elevated in the kidneys of Tsc1-KO ( n = 6) compared to WT ( n = 8) mice at 28 days. Image J analysis of TSC2 on day 45 indicates that all 3 sites are significantly more phosphorylated ( n = 8; 664Ser p-TSC2, p = 5.8533 × 10 −5 ; 939Ser p-TSC2, p = 0.032170819; and 1798Ser p-TSC2, p = 0.0197) in Tsc1-KO ( n = 8) compared to WT ( n = 8) mice. Results are presented as average ± SD. .

Article Snippet: The following antibodies were used for Western blot experiments: (1) tyr719 p-c-KIT (1:1000) (Cell Signaling Technology, Danvers, MA); (2) ser664 p-TSC2 (1:1000) (Invitrogen, Rockford, IL); (3) ser939 p-TSC2 (1:1000) (Cell Signaling Technology, Danvers, MA); (4) β-actin (1:1000) (Santa Cruz Biotechnology, Dallas, TX); (5) TSC1 (1:2000) (Protein Tech, Rosemont, IL); (6) TSC2 (1:2000) (Protein Tech, Rosemont, IL); (7) ERK1/2--WB (1:1000) (Cell Signaling Technology, Danvers, MA); (8) RSK1 WB (1:1000) (Cell Signaling Technology, Danvers, MA); (9) AKT WB (1:1000) (Cell Signaling Technology, Danvers, MA); and (10) S6 ribosomal (1:1000) (Cell Signaling Technology, Danvers, MA).

Techniques: Phospho-proteomics, Immunohistochemical staining, Labeling

Journal: EMBO Molecular Medicine

Article Title: The critical role of the proto-oncogene c- Kit in TSC renal cystogenesis

doi: 10.1038/s44321-025-00360-x

Figure Lengend Snippet: ( A ) Levels of phosphorylated and total ERK1/2, RSK1 and AKT were compared in WT ( n = 6), Tsc1-KO ( n = 6), and Tsc1/c-Kit-dKO ( n = 6) mice. Image J analysis of phosphorylated to total activated kinases indicates that the Tsc1- KO mice have significantly elevated levels of AKT ( p = 0.010332); and RSK1 ( p = 0.00378008) compared to Tsc1/c-Kit - dKO mice; while comparing the phosphorylated to total activated kinase levels in Tsc1 - KO and WT mice denotes significant changes in AKT ( p = 0.024352) and RSK1 ( p = 0.006182228) content. Results are presented as average ± SD. ( B ) Immunohistochemical labeling of p-ERK1/2, p-RSK1, and p-AKT in WT, Tsc1-KO , and Tsc1/c-Kit-dKO mice. “C” represents a cyst. Scale bar equals 20 μm. ( C ) Comparison of the renal content of phosphorylated TSC2 in WT ( n = 6), Tsc1-KO ( n = 6), and Tsc1/c-Kit- dKO ( n = 6) mice was conducted by western blot followed by Image J analysis. Quantitative analysis of the results indicates that the Tsc1-KO mice have significantly elevated levels of 644Ser p-TSC2 ( p = 0.004555628), 939Ser p-TSC2 ( p = 0.001481909), and 1798Ser p-TSC2 ( p = 0.010024844) compared to Tsc1/c-Kit - dKO mice; while comparing the phosphorylated to total activated TSC1 levels in Tsc1 - KO and WT mice indicates significant changes in 644Ser p-TSC2 ( p = 0.012737439) and 939Ser p-TSC2 ( p = 0.010559358) content. All statistics of Image J results were conducted using T-test analysis. Results are presented as average ± SD. .

Article Snippet: The following antibodies were used for Western blot experiments: (1) tyr719 p-c-KIT (1:1000) (Cell Signaling Technology, Danvers, MA); (2) ser664 p-TSC2 (1:1000) (Invitrogen, Rockford, IL); (3) ser939 p-TSC2 (1:1000) (Cell Signaling Technology, Danvers, MA); (4) β-actin (1:1000) (Santa Cruz Biotechnology, Dallas, TX); (5) TSC1 (1:2000) (Protein Tech, Rosemont, IL); (6) TSC2 (1:2000) (Protein Tech, Rosemont, IL); (7) ERK1/2--WB (1:1000) (Cell Signaling Technology, Danvers, MA); (8) RSK1 WB (1:1000) (Cell Signaling Technology, Danvers, MA); (9) AKT WB (1:1000) (Cell Signaling Technology, Danvers, MA); and (10) S6 ribosomal (1:1000) (Cell Signaling Technology, Danvers, MA).

Techniques: Immunohistochemical staining, Labeling, Comparison, Western Blot

Journal: EMBO Molecular Medicine

Article Title: The critical role of the proto-oncogene c- Kit in TSC renal cystogenesis

doi: 10.1038/s44321-025-00360-x

Figure Lengend Snippet: The receptor tyrosine kinase, c-KIT, is expressed on the basolateral aspect of A-IC. Activation of c-KIT leads to the potentiation of the RAS/ERK, PI3K/AKT, and RSK1 signal transduction pathways. These kinases phosphorylate and down-regulate TSC2 protein’s GTPase activating function; this leads to increased stability of GTP-RHEB, unregulated mTORC1 activity, and unchecked cell proliferation. Stem cell growth factor (SCF); SLC4A1 (AE1).

Article Snippet: The following antibodies were used for Western blot experiments: (1) tyr719 p-c-KIT (1:1000) (Cell Signaling Technology, Danvers, MA); (2) ser664 p-TSC2 (1:1000) (Invitrogen, Rockford, IL); (3) ser939 p-TSC2 (1:1000) (Cell Signaling Technology, Danvers, MA); (4) β-actin (1:1000) (Santa Cruz Biotechnology, Dallas, TX); (5) TSC1 (1:2000) (Protein Tech, Rosemont, IL); (6) TSC2 (1:2000) (Protein Tech, Rosemont, IL); (7) ERK1/2--WB (1:1000) (Cell Signaling Technology, Danvers, MA); (8) RSK1 WB (1:1000) (Cell Signaling Technology, Danvers, MA); (9) AKT WB (1:1000) (Cell Signaling Technology, Danvers, MA); and (10) S6 ribosomal (1:1000) (Cell Signaling Technology, Danvers, MA).

Techniques: Activation Assay, Transduction, Activity Assay

Journal: Cell reports

Article Title: HUWE1 stimulates mTORC1 activity by enhancing Rheb interaction with mTORC1 and supports de novo pyrimidine synthesis

doi: 10.1016/j.celrep.2025.116382

Figure Lengend Snippet: (A) HUWE1 is required for the Rheb G18A mutant to interact with CAD and mTOR. HEK293T cells expressing control, HUWE1, or CAD shRNA were transfected with the FLAG-Rheb S16H or FLAG-Rheb G18A mutant. FLAG-Rhebs were IPed, and levels of coIPed endogenous HUWE1, CAD, and mTOR were monitored. (B) HUWE1, but not CAD, is required for endogenous Rheb to interact with mTOR. Endogenous Rheb was IPed from MEFs expressing control, CAD, or HUWE1 shRNA under in vivo crosslinking conditions. Levels of coIPed endogenous mTOR and TSC2 were monitored. (C) Ablation of HUWE1 decreases Rheb ubiquitination. HUWE1 is knocked down with the shRNA in HEK293T cells expressing FLAG-tagged wild-type Rheb and G18A Rheb mutant. Levels of ubiquitinated Rheb were monitored using pan-ubiquitin (FK2) or K48-linked ubiquitin antibodies. (D and E) Ablation of HUWE1 decreases endogenous Rheb ubiquitination. HUWE1 is knocked down with the shRNA in HEK293T cells (D) and HepG2 cells (E). Levels of ubiquitinated endogenous Rheb (Ub-Rheb) were monitored. Quantification was displayed as a ratio of Ub-Rheb/non-Ub-Rheb. **** p < 0.0001, mean ± SEM, n = 3 (D) and p = 0.0768, mean ± SEM, n = 3 (E). Note that the KD efficiency of HUWE1 in HepG2 cells was low. (F) The E3 ligase activity of HUWE1 is required for Rheb to interact with mTOR. HEK293T cells ablated HUWE1 were transiently transfected with HA-GST-Rheb and FLAG-HUWE1 or FLAG-HUWE1 C4341S mutant. Cell lysates were subjected to a GST pull-down assay, and the levels of endogenous mTOR co-precipitated with HA-GST-Rheb were monitored. (G) Hypothetical model of HUWE1 function in forming Ub-Rheb-mTORC1-CAD complex. The HECT domain of HUWE1, which recognizes its substrates and ubiquitinated residues, and the CPSase domain of CAD are required for the interactions between HUWE1, CAD, and Rheb. The ablation of HUWE1 or its ubiquitin ligase activity disrupts the interaction between Rheb and mTOR or CAD. These observations suggest that HUWE1 plays a critical role in enhancing Rheb to interact with both mTORC1 and CAD, possibly through Rheb ubiquitin modification.

Article Snippet: Rabbit monoclonal anti- p -TSC2 T1462 (5B12) , Cell Signaling Technology , Cat#3617; RRID:AB_490956.

Techniques: Mutagenesis, Expressing, Control, shRNA, Transfection, In Vivo, Ubiquitin Proteomics, Activity Assay, Pull Down Assay, Modification