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2 protocols using tributyltin

1

Preparation of Chemical Stock Solutions

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Prochloraz (purity 99.1%, CAS 67747-09-5), tributyltin (TBT) chloride (96%, CAS 1461-22-9), tetrabromobisphenol A (TBBPA, 2,2-bis(3,5-dibromo-4-hydroxyphenyl)propane) (97%, CAS 79-94-7), perfluorooctanoic acid (PFOA) (96%, CAS 335-67-1), imazalil (99.7%, CAS 35554-44-0), butralin (99.8%, CAS 33629-47-9), clofibrate (CAS 637-07-0), and gemfibrozil (CAS 25812-30-0) were obtained from Sigma-Aldrich (St. Louis, MO). Tetrachlorobisphenol A (TCBPA, 2,2-bis(3,5-dichloro-4-hydroxyphenyl)propane) (95%, CAS 79-95-8) was purchased from TCI America (Portland, OR). All chemicals were dissolved and diluted in DMSO to make 1000× stock solutions.
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2

Molecular Probes and Antibodies for Cell Signaling

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Gramicidin, nigericin, tributyltin, valinomcycin, TMZ, and BMT were purchased from Sigma Chemicals (St. Louis, MO). Dulbecco’s Modified Eagle Medium (DMEM), accutase, goat anti-IgG secondary antibodies Alexa Fluor® 488 and 546, PBFI-AM, calcein-AM, MQAE, and pluronic acid were obtained from Invitrogen (Carlsbad, CA). Elite Vector Stain ABC System and 3-3′-diaminobenzidine (DAB) were from Vector Laboratories (Burlingame, CA). Rabbit anti-WNK1 (t-WNK1) (N-20, #sc-20468) was from Santa Cruz Biotechnology (Dallas, TX). Rabbit anti-phospho-WNK1 (p-WNK1) (T60, #AF4720) was from R&D Systems (Minneapolis, MN). Sheep anti-WNK3 (S156C) was developed previously described [35 (link)]. Mouse anti-NKCC (T4) antibody was from Developmental Studies Hybridoma Bank (Iowa City, IA). Mouse anti-α-tubulin (#2125), rabbit anti-ezrin (#3145), rabbit anti-ezrin/radixin/moesin (ERM) (#3142) and rabbit anti-phosphorylated-ERM (#3149) antibodies were from Cell Signaling (Beverly, MA). Rabbit anti-phosphorylated-NKCC1 (p-NKCC1) antibody (R5) was a kind gift from Dr. Biff Forbush (Yale University). Sheep anti-phosphorylated-NKCC1 recognizing the same residues (Thr212 and Thr 217) as R5 was developed as previously described [35 (link)]. Rabbit anti-SPAK/OSR1 (t-SPAK/t-OSR1) and rabbit anti-phosphorylated-SPAK (Ser373)/OSR1 (Ser325) (p-SPAK/p-OSR1) were developed as described before [36 (link),37 (link)].
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