Activity Assay:Article Title: Compositions and methods for the treatment of Lowe syndrome
Article Snippet: .. TABLE 1 Pharmacological agents used Compound Activity Source Catalog number Use (conc/time) calpeptin Rho activator Cytoskeleton, Inc CN01 137.5 μM/1 h C3 transferase Rho inhibitor Cytoskeleton, Inc CT04 10.5 nM/2 h (Fibroblasts), 4.2 nM/2 h (HK2) 4.2 nM/2 h (HK2) fasudil Rho kinase inhibitor Selleck Chemicals S1573 10 μM/3 h PF-3758309 PAK inhibitor AdooQ Bioscience A11930 1-10 μM/3-6 h ML7 MLCK inhibitor Enzo LifeSciences BML-EI197-0010 1 μM/6 h fluvastatin HMG-CoA Cayman Chemicals 10010337 1-100 μM/3-6 h simvastatin reductase inhibitors Cayman Chemicals 10010344 1-10 μM/8-12 h atorvastatin Toronto Research A791750 1-100 μM/3-6 h Chemicals pitavastatin Selleck Chemicals S1759 10 μM/12 h rosuvastatin Bio Vision 1955-5 100 μM/12 h or 10-20 μM/72 h (long-term exposure) FTI276 farnesylation inhibitor CalBioChem 344550 0.5 μM/6 h rapamycin mTOR inhibitor Cayman Chemicals No13346 100 nM/12 h or 10 nM/72 h (long-term exposure) WYE132 mTOR inhibitor BioVision Inc 2256-500 1 μM/72 h INK128 mTOR inhibitor MedKoo Bioscience 205495 20 nM/72 h SAR405 PI3K cIII inhibitor MedChem Express HY12481 1 μM/72 h TABLE 2 Antibodies Dilution used Antigen Host Source and applicationa mTOR (total) rabbit Cell Signaling (#2972) 1:1000 (WB) p-mTOR (S2448) rabbit Cell Signaling (#2971) 1:1000 (WB) p-mTOR (S2481) rabbit Cell Signaling (#2974) 1:1000 (WB) Akt (Total) rabbit Bioss (Bs-0115R) 1:500 (WB) p-Akt (S473) rabbit Cell Signaling (#4058) 1:1000 (WB) SGK-1 (Total) rabbit Millipore (# 07-315) 1:100 (WB) p-SGK-1 (S422) mouse Santa Cruz (sc-16745) 1:100 (WB) tubulin mouse Biolegend (627903) 1:500 (WB) acetylated tubulin mouse Sigma Aldrich (6-11B-1) 1:1000 (IIF) pericentrin rabbit Abcam (ab4448) 1:300 (IIF) vinculin mouse Sigma Aldrich (V9131) 1:800 (IIF) phospho-paxillin rabbit Epitomics (2128-1) 1:500 (WB) pan FAK rabbit Santa Cruz (C-20) 1:500 (WB) phospho-FAK rabbit Santa Cruz (11765-R) 1:50 (IIF), 1:500 (WB) LAMP2 mouse Santa Cruz (sc-18822) 1:150 (IIF) aWB: Western blotting; IIF: Indirect Immuno-Fluorescence. .. Cells and Culture Conditions Normal (GM07492) and LS primary dermal fibroblasts (GM01676 and GM03265) were obtained from the NIHGMS Human Genetic Cell Repository (Coriell Institute for Medical Research, Camden, NJ, USA).
Article Title: Compositions and methods for the treatment of Lowe Syndrome
Article Snippet: .. TABLE 1 Pharmacological agents used Compound Activity Source Catalog number Use (conc/time) calpeptin Rho activator Cytoskeleton, Inc CN01 137.5 μM/1 h C3 transferase Rho inhibitor Cytoskeleton, Inc CT04 10.5 nM/2 h (Fibroblasts), 4.2 nM/2 h (HK2) 4.2 nM/2 h (HK2) fasudil Rho kinase inhibitor Selleck Chemicals S1573 10 μM/3 h PF-3758309 PAK inhibitor AdooQ Bioscience A11930 1-10 μM/3-6 h ML7 MLCK inhibitor Enzo LifeSciences BML-EI197-0010 1 μM/6 h fluvastatin HMG-CoA Cayman Chemicals 10010337 1-100 μM/3-6 h simvastatin reductase inhibitors Cayman Chemicals 10010344 1-10 μM/8-12 h atorvastatin Toronto Research A791750 1-100 μM/3-6 h Chemicals pitavastatin Selleck Chemicals S1759 10 μM/12 h rosuvastatin Bio Vision 1955-5 100 μM/12 h or 10-20 μM/72 h (long-term exposure) FTI276 farnesylation inhibitor CalBioChem 344550 0.5 μM/6 h rapamycin mTOR inhibitor Cayman Chemicals No13346 100 nM/12 h or 10 nM/72 h (long-term exposure) WYE132 mTOR inhibitor BioVision Inc 2256-500 1 μM/72 h INK128 mTOR inhibitor MedKoo Bioscience 205495 20 nM/72 h SAR405 PI3K cIII inhibitor MedChem Express HY12481 1 μM/72 h TABLE 2 Antibodies Dilution used Antigen Host Source and applicationa mTOR (total) rabbit Cell Signaling (#2972) 1:1000 (WB) p-mTOR (S2448) rabbit Cell Signaling (#2971) 1:1000 (WB) p-mTOR (S2481) rabbit Cell Signaling (#2974) 1:1000 (WB) Akt (Total) rabbit Bioss (Bs-0115R) 1:500 (WB) p-Akt (S473) rabbit Cell Signaling (#4058) 1:1000 (WB) SGK-1 (Total) rabbit Millipore (# 07-315) 1:100 (WB) p-SGK-1 (S422) mouse Santa Cruz (sc-16745) 1:100 (WB) tubulin mouse Biolegend (627903) 1:500 (WB) acetylated tubulin mouse Sigma Aldrich (6-11B-1) 1:1000 (IIF) pericentrin rabbit Abcam (ab4448) 1:300 (IIF) vinculin mouse Sigma Aldrich (V9131) 1:800 (IIF) phospho-paxillin rabbit Epitomics (2128-1) 1:500 (WB) pan FAK rabbit Santa Cruz (C-20) 1:500 (WB) phospho-FAK rabbit Santa Cruz (11765-R) 1:50 (IIF), 1:500 (WB) LAMP2 mouse Santa Cruz (sc-18822) 1:150 (IIF) aWB: Western blotting; IIF: Indirect Immuno-Fluorescence. .. Normal (GM07492) and LS primary dermal fibroblasts (GM01676 and GM03265) were obtained from the NIHGMS Human Genetic Cell Repository (Coriell Institute for Medical Research, Camden, NJ, USA).
Western Blot:Article Title: Compositions and methods for the treatment of Lowe syndrome
Article Snippet: .. TABLE 1 Pharmacological agents used Compound Activity Source Catalog number Use (conc/time) calpeptin Rho activator Cytoskeleton, Inc CN01 137.5 μM/1 h C3 transferase Rho inhibitor Cytoskeleton, Inc CT04 10.5 nM/2 h (Fibroblasts), 4.2 nM/2 h (HK2) 4.2 nM/2 h (HK2) fasudil Rho kinase inhibitor Selleck Chemicals S1573 10 μM/3 h PF-3758309 PAK inhibitor AdooQ Bioscience A11930 1-10 μM/3-6 h ML7 MLCK inhibitor Enzo LifeSciences BML-EI197-0010 1 μM/6 h fluvastatin HMG-CoA Cayman Chemicals 10010337 1-100 μM/3-6 h simvastatin reductase inhibitors Cayman Chemicals 10010344 1-10 μM/8-12 h atorvastatin Toronto Research A791750 1-100 μM/3-6 h Chemicals pitavastatin Selleck Chemicals S1759 10 μM/12 h rosuvastatin Bio Vision 1955-5 100 μM/12 h or 10-20 μM/72 h (long-term exposure) FTI276 farnesylation inhibitor CalBioChem 344550 0.5 μM/6 h rapamycin mTOR inhibitor Cayman Chemicals No13346 100 nM/12 h or 10 nM/72 h (long-term exposure) WYE132 mTOR inhibitor BioVision Inc 2256-500 1 μM/72 h INK128 mTOR inhibitor MedKoo Bioscience 205495 20 nM/72 h SAR405 PI3K cIII inhibitor MedChem Express HY12481 1 μM/72 h TABLE 2 Antibodies Dilution used Antigen Host Source and applicationa mTOR (total) rabbit Cell Signaling (#2972) 1:1000 (WB) p-mTOR (S2448) rabbit Cell Signaling (#2971) 1:1000 (WB) p-mTOR (S2481) rabbit Cell Signaling (#2974) 1:1000 (WB) Akt (Total) rabbit Bioss (Bs-0115R) 1:500 (WB) p-Akt (S473) rabbit Cell Signaling (#4058) 1:1000 (WB) SGK-1 (Total) rabbit Millipore (# 07-315) 1:100 (WB) p-SGK-1 (S422) mouse Santa Cruz (sc-16745) 1:100 (WB) tubulin mouse Biolegend (627903) 1:500 (WB) acetylated tubulin mouse Sigma Aldrich (6-11B-1) 1:1000 (IIF) pericentrin rabbit Abcam (ab4448) 1:300 (IIF) vinculin mouse Sigma Aldrich (V9131) 1:800 (IIF) phospho-paxillin rabbit Epitomics (2128-1) 1:500 (WB) pan FAK rabbit Santa Cruz (C-20) 1:500 (WB) phospho-FAK rabbit Santa Cruz (11765-R) 1:50 (IIF), 1:500 (WB) LAMP2 mouse Santa Cruz (sc-18822) 1:150 (IIF) aWB: Western blotting; IIF: Indirect Immuno-Fluorescence. .. Cells and Culture Conditions Normal (GM07492) and LS primary dermal fibroblasts (GM01676 and GM03265) were obtained from the NIHGMS Human Genetic Cell Repository (Coriell Institute for Medical Research, Camden, NJ, USA).
Article Title: Compositions and methods for the treatment of Lowe Syndrome
Article Snippet: .. TABLE 1 Pharmacological agents used Compound Activity Source Catalog number Use (conc/time) calpeptin Rho activator Cytoskeleton, Inc CN01 137.5 μM/1 h C3 transferase Rho inhibitor Cytoskeleton, Inc CT04 10.5 nM/2 h (Fibroblasts), 4.2 nM/2 h (HK2) 4.2 nM/2 h (HK2) fasudil Rho kinase inhibitor Selleck Chemicals S1573 10 μM/3 h PF-3758309 PAK inhibitor AdooQ Bioscience A11930 1-10 μM/3-6 h ML7 MLCK inhibitor Enzo LifeSciences BML-EI197-0010 1 μM/6 h fluvastatin HMG-CoA Cayman Chemicals 10010337 1-100 μM/3-6 h simvastatin reductase inhibitors Cayman Chemicals 10010344 1-10 μM/8-12 h atorvastatin Toronto Research A791750 1-100 μM/3-6 h Chemicals pitavastatin Selleck Chemicals S1759 10 μM/12 h rosuvastatin Bio Vision 1955-5 100 μM/12 h or 10-20 μM/72 h (long-term exposure) FTI276 farnesylation inhibitor CalBioChem 344550 0.5 μM/6 h rapamycin mTOR inhibitor Cayman Chemicals No13346 100 nM/12 h or 10 nM/72 h (long-term exposure) WYE132 mTOR inhibitor BioVision Inc 2256-500 1 μM/72 h INK128 mTOR inhibitor MedKoo Bioscience 205495 20 nM/72 h SAR405 PI3K cIII inhibitor MedChem Express HY12481 1 μM/72 h TABLE 2 Antibodies Dilution used Antigen Host Source and applicationa mTOR (total) rabbit Cell Signaling (#2972) 1:1000 (WB) p-mTOR (S2448) rabbit Cell Signaling (#2971) 1:1000 (WB) p-mTOR (S2481) rabbit Cell Signaling (#2974) 1:1000 (WB) Akt (Total) rabbit Bioss (Bs-0115R) 1:500 (WB) p-Akt (S473) rabbit Cell Signaling (#4058) 1:1000 (WB) SGK-1 (Total) rabbit Millipore (# 07-315) 1:100 (WB) p-SGK-1 (S422) mouse Santa Cruz (sc-16745) 1:100 (WB) tubulin mouse Biolegend (627903) 1:500 (WB) acetylated tubulin mouse Sigma Aldrich (6-11B-1) 1:1000 (IIF) pericentrin rabbit Abcam (ab4448) 1:300 (IIF) vinculin mouse Sigma Aldrich (V9131) 1:800 (IIF) phospho-paxillin rabbit Epitomics (2128-1) 1:500 (WB) pan FAK rabbit Santa Cruz (C-20) 1:500 (WB) phospho-FAK rabbit Santa Cruz (11765-R) 1:50 (IIF), 1:500 (WB) LAMP2 mouse Santa Cruz (sc-18822) 1:150 (IIF) aWB: Western blotting; IIF: Indirect Immuno-Fluorescence. .. Normal (GM07492) and LS primary dermal fibroblasts (GM01676 and GM03265) were obtained from the NIHGMS Human Genetic Cell Repository (Coriell Institute for Medical Research, Camden, NJ, USA).
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