Brefeldin A (BFA), cycloheximide, tyrphostin A23 (TyrA23), wortmannin, and concanamycin A (ConcA) were obtained from Sigma-Aldrich and used from stock solutions in DMSO (50 mM BFA, cycloheximide, TyrA23; 30 mM wortmannin, 2 mM ConcA). FM4-64 was purchased from Molecular Probes (2 mM stock solution in water). Five-day-old seedlings were incubated for the indicated times in liquid 1/2 MS medium containing 50 µM BFA, 50 µM cycloheximide, 75 µM TyrA23, 33 µM wortmannin, and 2 µM ConcA. For FM4-64 staining seedlings were incubated in 4 µM FM4-64 in liquid 1/2 MS medium for 5 min prior to imaging. 4-Hydroxytamoxifen was obtained from Sigma-Aldrich (10 mM stock solution in ethanol). Seedlings were grown for 3 d on 1/2 MS plates, transferred onto 1/2 MS plates containing 2 µM 4-Hydroxytamoxifen (or ethanol as mock treatment) for 4 d and then imaged using confocal microscopy.
Tyrphostin a23
Manufactured by Merck Group
Tyrphostin A23 is a synthetic compound that functions as a tyrosine kinase inhibitor. It is commonly used in biological research applications to study cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Cycloheximide (chx), by Merck Group (2 mentions)
Fm4 64, by Thermo Fisher Scientific (1 mentions)
4 hydroxytamoxifen, by Merck Group (1 mentions)
Wortmannin, by Merck Group (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Concanamycin a, by Merck Group (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Jsh 23, by Merck Group (1 mentions)
Cycloheximide (chx), by Cayman Chemical (1 mentions)
Phluoroglucinol, by Merck Group (1 mentions)
Actinomycin d, by Bio-Techne (1 mentions)
Fm4 64 dye, by Thermo Fisher Scientific (1 mentions)
Tyra23, by Merck Group (1 mentions)
Wortmannin wm, by Merck Group (1 mentions)
Concanamycin a cona, by Merck Group (1 mentions)
Sortin 2, by ChemBridge (1 mentions)
5 protocols using tyrphostin a23
1
Organelle Dynamics Modulation Assay
2
Phagocytosis Assay for Microglia
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Bead phagocytosis assays were carried out as described in Lian et al.34 . Briefly, green fluorescent latex beads were opsonized in FBS for 1 h at 37 °C at a ratio of 1:5 beads to FBS. This solution was then diluted into complete differentiation medium to a final concentration of 0.01% (v/v) beads. MG were treated with PBS (control), 1 µM N1 or 10 µM tyrphostin-A23 (Sigma-Aldrich) for 1 hour before exchanging the media for the bead-containing media. MG were incubated for a further hour with the beads before washing with cold PBS and fixing in 4% (v/v) paraformaldehyde. The number of cells showing uptake of the fluorescent beads was counted in three or more fields of 50–100 cells per independent repeat.
3
Agrobacterium-Mediated Transient Assays
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A. tumefaciens cells were grown, and the cell concentrations were adjusted to OD600 = 0.5 in H2O; ES1 was added into the cell suspensions at a final concentration of 25 μM. The mixtures were then infiltrated into N. benthamiana leaves. As the control, A. tumefaciens cell suspensions in H2O were infiltrated into N. benthamiana leaves. The infiltrated plants were then placed at 22°C under a 16-hour light/8-hour dark photoperiod.
During a stable transformation assay, A. thaliana roots from individual seedlings were cut into 3- to 5-mm segments and mixed with ES1 or tyrphostin A23 (Sigma) at a final concentration of 60 or 50 μM, respectively, in H2O, and the mixtures were then kept in the dark for 3 hours. As the control, the root fragments were treated with H2O alone. The root fragments were then mixed with A. tumefaciens for root transformation assays, as described above.
During a stable transformation assay, A. thaliana roots from individual seedlings were cut into 3- to 5-mm segments and mixed with ES1 or tyrphostin A23 (Sigma) at a final concentration of 60 or 50 μM, respectively, in H2O, and the mixtures were then kept in the dark for 3 hours. As the control, the root fragments were treated with H2O alone. The root fragments were then mixed with A. tumefaciens for root transformation assays, as described above.
4
Inhibitors for Imaging Sulfenic Acid
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The following inhibitors and concentrations were added 2 h prior to imaging (unless stated otherwise in the text) and kept in the solution during imaging unless otherwise stated: DMSO vehicle (0.15%); Phluoroglucinol (25 µM; Sigma-Aldrich, Cat. no. 79330); 5,5-dimethyl-1,3-cyclohexanedion, dimedone (1.5 mM; Sigma-Aldrich, Cat. no. D153303). Dimedone selectively and irreversibly binds to sulfenic acid (26 (link)) and was added 1 h prior to and 30 min after amputation. Click-Tag DYn-2 (10 µM; Cayman Chemical, Cat. no. 11220); EGFR inhibitor (8 and 10 µM; Cayman Chemical, Cat. no. 16363); Erbstatin (10 µM; Cayman Chemical, Cat. no.10010238); Lavendustin A (10 µM; EMD Millipore, Cat. no. 428150); Tyrphostin A23 (10 µM; Sigma-Aldrich, Cat. no. T7165); actinomycin D (10 µM; Tocris, Cat. no. 1229); 6-MP (10 µM; Sigma-Aldrich, Cat. no. 38171); DRB (10 µM; Cayman Chemical, Cat. no. 10010302); cycloheximide (10 µM; Cayman Chemical, Cat. no. 14126); hydrogen peroxide (0.01% and 0.3%; Sigma-Aldrich, Cat. no. H1009); GM6001 (10 µM; Ilomastat, Millipore Sigma, Cat. no. CC1100); DB04760 (MMP-13 inhibitor, Millipore Sigma, Cat. No. 444283); NF-κB inhibitor (10 µM; JSH-23, Sigma-Aldrich, Cat. no. J4455). MMP-9 inhibitor I (Cayman Chemical, Cat. no.15942).
5
Inhibitor Infiltration in Transient Transformation
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All the drug treatments were performed by inhibitor infiltration in transiently transformed leaf. The final concentration of the inhibitor used were: 350 μM for tyrphostin A23 (TyrA23; Sigma-Aldrich2 ), 100 μM for salycid acid (SA; Sigma-Aldrich2 ), 2 μM for concanamycin A (ConA; Sigma-Aldrich2 ), 3 μM for wortmannin (Wm; Sigma-Aldrich2 ), 20 μM for Sortin 2 (ChemBridge Corporation3 ), 50 μM for Endosidin 5 (ES5; ChemBridge Corporation3 ).
To test BFA (Sigma-Aldrich2 ) effect on endocytosis it was necessary to distinguish between newly synthesized and endocytosed proteins, so leaves transiently expressing PGIP2-GFP and stably expressing secGFP-CesA6 were infiltrated with cycloheximide (CHX; Sigma-Aldrich2 ) to the final concentration of 300 μM. The same procedure was applied to ConA treatment on secGFP-CesA6 accordingly to Dettmer et al. (2006) (link). The leaf discs (1 cm diameter) taken from treated leaves were incubated by immersion in water containing 10 μM of FM4-64 dye (Invitrogen Molecular Probes4 ) before treatment with BFA. In the case of co-expression of secGFP-CesA6 with ST52-mCherry, CHX pre-treatment was not applied. Leaf discs were treated by immersion in solution with BFA at the final concentration of 100 μM and the induced fluorescent pattern was compared to the control conditions. Images were collected at the indicated times of incubation.
To test BFA (Sigma-Aldrich
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