The following antibodies were used: anti-P2RX7 (Synaptic Systems, 177003) 1:1000, anti-MAPK1-MAPK3 (Cell Signaling Technology, 9102) 1:2000, anti-phospho-MAPK1-MAPK3 (Cell Signaling Technology, 9106) 1:1000, anti-ACTB (Sigma, A2066) 1:1000, anti-LC3B (Sigma, L7543) 1:1000, anti-GAPDH (Sigma, G9545) 1:1000, anti-COX4/COXIV (Abcam, ab14744) 1:000, anti-BECN1 (Santa Cruz Biotechnology, 48381) 1:200. Other chemicals used were as follows: P2RX7 antagonists A438079 and A804598 (Tocris Bioscience, 2972 and 4473, respectively) and Brilliant Blue G (BBG, Ascent Scientific, ASC-389), HSPA2 and HSP90 inhibitors, VER155008, geldanamycin and 17-DMAG (Tocris Biosciences, 3803, 1368 and 2610, respectively), Proteostat aggresome detection kit (Enzo Life Sciences, ENZ-51035-K100), HSPA2 inhibitor KNK437 (Merck Millipore, 373260), the cell permeable, fluorogenic CASP3-CASP7 substrate DEVD-Nucview488 (Cambridge Biosciences, BT10403) and the mitochondrial membrane potential-sensitive probe tetramethylrhodamine ethyl ester (TMRE; Sigma, 87917), digoxin and digitoxigenin (Sigma, D6770 and D9404, respectively).
17 dmag
Manufactured by Bio-Techne
Sourced in United Kingdom
17-DMAG is a heat shock protein 90 (Hsp90) inhibitor. It functions by binding to and inhibiting the Hsp90 chaperone protein, which is involved in the folding and stabilization of various client proteins.
Automatically generated - may contain errors
Lab products found in correlation
Digitoxigenin, by Merck Group (1 mentions)
Anti lc3b, by Merck Group (1 mentions)
A438079, by Bio-Techne (1 mentions)
Proteostat aggresome detection kit, by Enzo Life Sciences (1 mentions)
Anti actb, by Merck Group (1 mentions)
Anti becn1, by Santa Cruz Biotechnology (1 mentions)
Geldanamycin, by Bio-Techne (1 mentions)
Ver155008, by Bio-Techne (1 mentions)
Knk437, by Merck Group (1 mentions)
A804598, by Bio-Techne (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Oligomycin, by Bio-Techne (1 mentions)
Doxycycline monohydrate, by Merck Group (1 mentions)
Dorsomorphin dihydrochloride, by Bio-Techne (1 mentions)
Dorsomorphin, by Bio-Techne (1 mentions)
Aicar, by Bio-Techne (1 mentions)
Etomoxir, by Bio-Techne (1 mentions)
Akti 1 2, by Bio-Techne (1 mentions)
Sodium salicylate, by Merck Group (1 mentions)
Trypan blue exclusion assay, by Thermo Fisher Scientific (1 mentions)
3 protocols using 17 dmag
1
Autophagy Regulation by P2RX7 Signaling
2
Small Molecule Inhibitor Protocol
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Sodium salicylate (CAS N 54-21-7), oligomycin from Streptomyces diastatochromogenes (CAS N 1404-19-9) and doxycycline monohydrate (CAS N 17086-28-1) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 17-DMAG (CAS N150270-08-9) was purchased from Tebu-bio (Barcelona, Spain). AICAR (CAS N 2627-69-2), Akti-1/2 (CAS N 612847-09-3), dorsomorphin dihydrochloride (CAS N 1219168-18-9), SC79 (CAS N 305834-79-1), and (R)-(+)-etomoxir sodium salt (CAS N 828934-41-4) were purchased from TOCRIS (Bristol, UK).
AICAR, Akti-1/2, dorsomorphin, etomoxir, and SC79 stock dilutions were prepared in DMSO at 20 mM whereas oligomycin and 17-DMAG were prepared at 2 and 1 mmol L−1, respectively. The drug Sodium salicylate was prepared before use in the appropriate culture media at a starting concentration of 200 µmol L−1.
AICAR, Akti-1/2, dorsomorphin, etomoxir, and SC79 stock dilutions were prepared in DMSO at 20 mM whereas oligomycin and 17-DMAG were prepared at 2 and 1 mmol L−1, respectively. The drug Sodium salicylate was prepared before use in the appropriate culture media at a starting concentration of 200 µmol L−1.
3
Aortic VSMC Viability Assay
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Materials. Smooth muscle cell growth and basal medium were purchased from cell applications, inc. (San diego, ca). Trypsin and trypsin neutralizing solution were purchased from cell applications inc. (San diego, ca). 17-aaG and 17-dMaG were purchased from Tocris Bioscience (Bristol, uK). Fibronectin and PdGF were purchased from r&d Systems (Minneapolis, Mn).
Cell culture. Human aortic VSMcs (cell applications, inc., San diego, ca, uSa) were used in early passage (P3-P5). cells were made quiescent by incubation in serum free media (SFM) for 48 h at 37˚c in a tissue culture incubator (5% co 2 ).
Cell viability. cell viability was determined with the Trypan Blue exclusion assay (Thermo Fisher Scientific, Waltham, MA) using the Countess Cell counter (Thermo Fisher Scientific, Waltham, Ma) according to the manufacturer's instructions. all experiments were performed in triplicate studying 17-aaG or 17-dMaG at three different concentrations (100 nM, 10 or 30 µM). The doses chosen have been previously shown to be effective (13) .
Cell culture. Human aortic VSMcs (cell applications, inc., San diego, ca, uSa) were used in early passage (P3-P5). cells were made quiescent by incubation in serum free media (SFM) for 48 h at 37˚c in a tissue culture incubator (5% co 2 ).
Cell viability. cell viability was determined with the Trypan Blue exclusion assay (Thermo Fisher Scientific, Waltham, MA) using the Countess Cell counter (Thermo Fisher Scientific, Waltham, Ma) according to the manufacturer's instructions. all experiments were performed in triplicate studying 17-aaG or 17-dMaG at three different concentrations (100 nM, 10 or 30 µM). The doses chosen have been previously shown to be effective (13) .
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