Notch-C-20, Hes5 (Santa Cruz); hamartin, p53 (1C12), phospho-S6, phospho-S6K (T389), Hes1 and Gapdh (Cell Signaling); Nodal (Abnova). Secondary steps for IHC used a detection kit (Invitrogen). Secondary antibodies conjugated with fluorochromes (AF488) and DAPI were used for immunofluorescence. Images were captured with Nikon TE2000 microscope and analyzed using Nikon Elements Advanced Research software. In addition, Aperio digital pathology system (currently Leica Biosystems, IL, USA) algorithm-based software was used to locate and quantitate staining.
Hamartin
Manufactured by Cell Signaling Technology
Hamartin is a protein that plays a crucial role in cellular signaling pathways. It functions as a tumor suppressor and is involved in the regulation of cell growth, proliferation, and survival. Hamartin is a key component of the tuberous sclerosis complex (TSC), which acts as a negative regulator of the mTOR (mammalian target of rapamycin) signaling pathway, a central hub for cellular metabolism and growth.
Automatically generated - may contain errors
Lab products found in correlation
Ps6 s240 244, by Cell Signaling Technology (2 mentions)
Ps6 s235 236, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Tissuelyser, by Qiagen (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Aperio digital pathology system, by Leica (1 mentions)
Imagequant software, by GE Healthcare (1 mentions)
Gapdh, by GE Healthcare (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Criterion tgx gel, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Phosstop, by Roche (1 mentions)
Complete mini, by Roche (1 mentions)
4 protocols using hamartin
1
Immunofluorescence and IHC Quantitation Protocol
2
Immunoblotting Analysis of LV Tissue
3
Retinal Protein Extraction and Western Blotting
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The cornea, lens, ON and blood vessels were removed from enucleated eyes and the retina/RPE was snap-frozen in liquid nitrogen. Tissues were homogenized in SDS lysis buffer [100 mM Tris, pH 6.8; 2% (w/v) SDS], containing protease and phosphatase inhibitor cocktails (Complete Mini & PhosSTOP, Roche), using a TissueLyser (Qiagen Inc.). The protein extracts were centrifuged at 14,000 g for 5 min at 4°C and the concentration of the soluble fraction was measured using the BCA Protein Assay Kit (Fisher Thermo Scientific). Western blotting was performed on soluble protein extracts (10 µg) using Criterion TGX gels (Bio-Rad) and nitrocellulose membranes (Bio-Rad) as previously described (Mahmood and Yang, 2012 (link)). Reactive proteins were visualised using SuperSignal West Dura Extended Duration Substrate (Fisher Thermo Scientific). Imaging and quantification was performed using a ChemiDoc MP Imaging System (Bio-Rad). The following antibodies and dilutions were used: Hamartin (Cell Signaling Technology, 1:1000), S6 (Cell Signaling Technology, 1:1000), pS6 (S235/236) (Cell Signaling Technology, 1:1000), pS6 (S240/244) (Cell Signaling Technology, 1:1000) and GAPDH (Cell Signaling Technology, 1:30,000).
4
Western Blot Analysis of Protein Expression
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The lens was removed from enucleated eyes and the NR and RPE frozen in liquid nitrogen. Tissues were homogenised in SDS lysis buffer [100 mM Tris-HCl, pH 6.8; 2% (w/v) SDS] containing both protease and phosphatase inhibitors (Complete Mini & PhosSTOP, Roche) using a TissueLyser (Qiagen). The concentration of the soluble protein fraction was measured using the BCA Protein Assay Kit (Fisher Thermo Scientific). Immunoblotting was performed on protein extracts (6 µg) using Criterion TGX gels and nitrocellulose membranes (Bio-Rad) (Mahmood and Yang, 2012 (link)). Reactive proteins were visualised using SuperSignal West Dura Extended Duration Substrate (Fisher Thermo Scientific). Quantification was performed using a ChemiDoc MP Imaging System (Bio-Rad). The following antibodies and dilutions were used: Hamartin (1:1000, Cell Signaling Technology, 4906), S6 (1:2000, Cell Signaling Technology, 2217), pS6S235/236 (1:1000, Cell Signaling Technology, 4857), pS6S240/244 (1:2000, Cell Signaling Technology, 5364) and GAPDH (1:30,000, Cell Signaling Technology, 2118). All immunoblotting antibodies have been verified in a previous study (Jones et al., 2015 (link)).
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