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