General protocols for growth media and growth conditions for N. crassa have been previously described (Davis, 2000 ) and are also available at the Fungal Genetics Stock Center1 . Strains used in this study are listed in Table 1 . All strains, excluding MYC-PPH1, were grown in either liquid or solid (supplemented with 1.5% agar) Vogel’s minimal medium, with 1.5% (w/v) sucrose (VgS). The MYC-PPH1 strain was grown in liquid Vogel’s minimal medium with 0.1% glucose, supplemented with 0.17% arginine and 0.01 M quinic acid, at pH 5.8 (Cheng et al., 2001 (link)). When required, the medium was supplemented with 10 μg/ml hygromycin B (Duchefa), 100 mM cantharidin (Abcam Biochemicals) or 100 μg/ml L -histidine (Sigma–Aldrich).
Cantharidin
Manufactured by Abcam
Cantharidin is a naturally occurring chemical compound found in certain insects, particularly blister beetles. It is a potent skin irritant and has been used in various medical and scientific applications. Cantharidin's core function is as a topical agent, with potential uses in research and clinical settings.
Automatically generated - may contain errors
Lab products found in correlation
L histidine, by Merck Group (1 mentions)
Hygromycin b, by Duchefa Biochemie (1 mentions)
Nonimmune rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
Clone 2g10, by Merck Group (1 mentions)
Epr5479, by Abcam (1 mentions)
2 protocols using cantharidin
1
N. crassa Growth Conditions and Media
2
Characterization of Phosphorylated GluA1 Antibodies
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Cantharidin (Abcam) was prepared as a 20 mM stock solution in DMSO. All other chemicals were obtained from Sigma-Aldrich. Phospho-T840 (1:2,000) and total GluA1 antibodies (1:2000, rabbit monoclonal EPR5479) were obtained from Abcam and the phospho-S845 antibody (1:1000) was obtained from Millipore. Monoclonal antibodies against a neuronal specific isoform (βIII) of tubulin (1:20,000, Clone 2G10) and AMPAR GluA2 subunits (1:1000-500, Clone N52A/42) were obtained from Sigma-Aldrich and the UC Davis/NIH NeuroMab facility, respectively. Control immunoprecipitations were performed using a nonimmune rabbit IgG obtained from Santa Cruz Biotechnology. Horseradish peroxidase conjugated secondary antibodies (1:2,000) were obtained from GE Healthcare. The phospho-T840 and phospho-S845 GluA1 antibodies used in these experiments have been extensively characterized by us (Delgado et al. 2007 (link); Gray et al. 2014 (link)) and others (Hosokawa et al. 2015 (link); Toda and Huganir 2015 (link)) and do not recognize nonphosphorylated GluA1 or GluA1 phosphorylated at other sites.
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