Protocols





Agrobacterium Competent Cells

BL21(DE3) induction

Competent E. coli BL21(DE3)

Dipping protocol for A. thaliana

Gateway Cloning

Gel Electrophoresis of DNA

In vitro pollen germination
MC1061 E. coli competent cells

Minipreps

N. tabacum transient transformation 1 

N. tabacum transient transformation 2

Protein interactions



Fluorescent Probes


-Calcium detection
Arabidopsis plants are incubated in Eppendorf tubes contaning 20 μm Fluo-3 AM in water and  incubated for 1 h. For imaging, plants were washed and observed.

-Sodium detection
Roots are incubated in Eppendorf tubes in 500 µL of the 10 mM Sodium Green solution (S-6901, Molecular Probes, Eugene, OR, USA).After 1 h of incubation, the samples were examined using confocal microscopy

-NO detection
Seedlings are incubated for 30 min in freshly prepared 15 mM DAF. Then samples are washed and observed.

-Propidium Iodide
Arabidopsis seedling roots are stained with 5-10 µg/mL of propidium iodide for 20 to 30 min.

-FM 4-64 (Molecular Probes)
Arabidopsis leaves are submerged in 8.2 µM FM4-64  in water for 15 min, then leaf are rinsed in water and observed

-DIOC6
endomembranes are stained with DiOC6 (Molecular Probes; working solution: 1.8 mM) in water for 30 min.

-Rhodamine B
 ER staining with Rhodamine B hexyl ester solution (1.6 μM) for 10 minutes.

-DAPI staining 
the seedlings were immerged in 1 μg/ml (w:v) DAPI (Sigma) in 1x PBS buffer with 1% (v : v) Triton X- 100 for 10 min at room temperature, followed by washing with PBS.

-ROS detection
Roots were incubated in 10 μM DCFH-DA  in 1x PBS buffer for 30 min and washed three times in PBS buffer

Drug Treatments

-BFA
A sample is inserted in an eppendorf tube containing a solution of tap water and brefeldin A to a final concentration of 100 μg/ml. The sample is kept in this solution for 1 hour, protected from the light. When the incubation time is over the sample is poured on a microscope slide and observed.

-Latrunculin B (F-actin inhibitor)
25 µM (Calbiochem; stock solution, 10 mM in DMSO), for 1 hour.

-Wortmannin
wortmannin solution (stock solution at 1 mM in DMSO) is added to a final concentration of 16.5 μM or 33 μM, followed by incubation for 1 h.


-tyrA23 (inhibitor of clathrin-mediated endocytosis)
Arabidopsis leaves are treated 1hour with 50 μM tyrA23.

-tyrphostin A51(inactive analogue of TyrA23)
Arabidopsis leaves are treated 1hour with 50 μM tyrA51.

-cytochalasin D (disrupts F-actin organization)
10 μM, 2 hours

-oryzalin (microtubule inhibitor)
seedlings are incubated with 10 µM oryzalin for 20 min

-2,3-butanedione monoxime (BDM; myosin inhibitor)
seedlings are incubated with 50 mM BDM for 20 min 

-propyzamide (tubulin-depolymerizing agent) 
10 µg/ml, 2 hours

-colchicine (tubulin-depolymerizing agent) 
10 mM, 2 hours


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