MG132 (Sigma-Aldrich)) was dissolved in DMSO to yield a 10 mM stock, then stored at −20° C until use. Doxycycline (Dox) (BD Biosciences) was dissolved in PBS to a 1 mg/ml stock. α-Flag agarose, α-Flag-HRP conjugated antibodies and α-β-actin were obtained from Sigma-Aldrich. α-VEGFR-1 antibodies were purchased from Epitomics, α-E6 N-terminus antibodies were obtained from Euromedex (France), α-E-cadherin was purchased from Cell Signalling Technology, α-caspase 8 antibodies were purchased from BD Biosciences, α-HA was obtained from Roche Applied Science, and secondary ImmunoPure Antibody HRP conjugated antibodies were obtained from Fisher Scientific. α-p53 p122 antibodies were purified from conditioned media obtained during hybridoma growth.
Doxycycline dox
Manufactured by BD
Sourced in United States
Doxycycline (DOX) is a broad-spectrum antibiotic belonging to the tetracycline class. It is a commonly used laboratory reagent for various research applications.
Automatically generated - may contain errors
Lab products found in correlation
α β actin, by Merck Group (1 mentions)
α flag agarose, by Merck Group (1 mentions)
α e cadherin, by Cell Signaling Technology (1 mentions)
Mg132, by Merck Group (1 mentions)
Z vad fmk, by Apexbio (1 mentions)
α sharpin, by Proteintech (1 mentions)
α hsp90, by Santa Cruz Biotechnology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Hygromycin, by Thermo Fisher Scientific (1 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Imagexpress micro, by Molecular Devices (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
5 protocols using doxycycline dox
1
MG132 and Doxycycline Reagents Protocol
2
DISC Lysis Buffer Protein Complex Study
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DISC lysis buffer (20 mM Tris–HCL pH7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X‐100, 10% Glycerol, H2O). The following reagents were used: human and mouse TNF (Enzo), zVAD‐FMK (Apex Bio), SM‐164 (gift from Shaomeng Wang), Ripk1i (GSK'963, gift from GlaxoSmithKline plc.), doxycycline (DOX) (BD Bioscience), Riboxxol (Riboxx Gmbh). The following antibodies were used: α‐RIPK1 (Cell Signaling; 3493), α‐CYLD (Cell Signaling; D1A10), α‐SHARPIN (Proteintech), α‐TRADD (BD Biosciences), α‐TNFR1 [H5] (Santa Cruz Biotechnology), α‐HSP90 (Santa Cruz Biotechnology), α‐CASP‐8—for Western blot (WB)—post‐immune‐precipitation (IP) (MBL), α‐CASP‐8—for IP [C‐20] (Santa Cruz Biotechnology), α‐Casp‐8 for rat (Cell Signalling; 9429), α‐ FADD for IP and WB [M‐19] (Santa Cruz Biotechnology), α‐Ripk3 (Pro‐science; 2283). All antibodies were used at a 1:1,000 dilution.
3
Culturing and Genetic Manipulation of C. albicans
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All C. albicans strains were archived in 25% glycerol and stored at -80°C. Overnight cultures were grown in YPD (1% yeast extract, 2% bactopeptone, 2% glucose) at 30°C. 2% agar was added for solid media. Strains were constructed according to standard protocols. Strain construction is described in S1 Text and strains used in this study are listed in S2 Table . Doxycycline (DOX, BD Biosciences #631311) was formulated at 20 mg/ml in water and used at a final concentration of 0.05 μg/ml as specified. 1-Naphthyl-PP1 (1-NA-PP1, Cayman Chemical #221243-82-9) was formulated at 5 mM in DMSO (Sigma-Aldrich) and used at a final concentration of 5 μM. Geldanamycin (GdA, Cedarlane, ant-gl-5) was formulated at 5 mM in DMSO and used at a final concentration of 10 μM. N6,2’-O-Dibutyryladenosine 3’, 5’-cyclic monophosphate sodium salt (dibutyryl cAMP, Sigma-Aldrich, D0627) was formulated at 100 mg/ml and used at a final concentration of 10 mg/ml.
4
Genetic Engineering of C. albicans Strains
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All C. albicans strains were archived in 25% glycerol and stored at −80 °C. Strains were grown in YPD (1% yeast extract, 2% bactopeptone, and 2% glucose) at 30 °C unless otherwise specified. For solid media, 2% agar was added. Strains were constructed according to standard protocols. Strains used in this study are listed in Supplementary Table 1 . Caspofungin was generously provided by Terry Roemer (Department of Infectious Diseases, Merck Research Laboratories) and was diluted to a 100 µg/ml stock, and used at a final concentration of 125 ng/ml. Doxycycline (DOX, BD Biosciences #631311) was formulated in a 20 mg/ml stock in water and used at a final concentration of 0.02 µg/ml or 1 µg/ml, as specified.
5
Tau Aggregation Inhibition Screening
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The ΔK280 tauRD-DsRed construct was used to establish Flp-In 293 cells and maintained according to the supplier’s instructions (Thermo Fisher Scientific). A medium containing 5 μg/mL of blasticidin and 100 μg/mL of hygromycin (Thermo Fisher Scientific) was used to grow these cells. ΔK280 tauRD-DsRed 293 cells were plated into 96-well (2×104/well) plates, grown for 24 hours, and pretreated with Congo red (1–20 μM) or TCM extracts (NH001–NH042; 100–500 μg/mL) for 8 hours. Doxycycline (Dox; 1 μg/mL, BD, Franklin Lakes, NJ, USA) was then added to the medium to induce tauRD-DsRed expression for 3 days. The cells were stained with Hoechst 33342 (0.1 μg/mL; Sigma-Aldrich Co). Images of the cells were automatically obtained and analyzed using a high-content analysis system (ImageXpress MICRO; Molecular Devices LLC, Sunnyvale, CA, USA), with excitation/emission wavelengths at 543/593 nm.
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