All experiments, unless otherwise specified, were performed in 10 [K.sup.+] artificial seawater (ASW; composition in millimoles: 430 NaC1
, 10 KCl, 50 Mg[C1.sub.2], 10 Ca[C1.sub.2], 10 HEPES, pH 7.5, 970 mOsm).
For this, the clots formed in vitro were washed exhaustively with high-salt buffers (0.5 M NaC1
, 10 mM [CaCl.sub.2], 10 mM Tris, pH 7.3) and detergent (0.5% Triton-X 100), and were then scraped from the petri dish and transferred to microcentrifuge tubes, where they were compressed by centrifugation, washed further, and finally eluted by removal of [Ca.sup.+2] with 0.1 M EDTA.
DNA-PK holoenzyme was immunoprecipitated with PAS beads that had been coated with the Ku antibody as follows: PAS beads were pre-swollen in kinase assay buffer (50 mM HEPES, pH 7.4; 10 mM EGTA, 40 mM NaC1
, 100 mM potassium acetate, 8.5 mM [CaCl.sub.2], 2.29 mM [MgCl.sub.2], 277 mM glycerol).
Binding at atmospheric pressure was determined in a volume of 100 [micro]1 The assay mixture contained 50 mM Tris HCl, pH 7.4 at the assay temperature of 5[degrees]C, 1 mM EDTA, 5 mM magnesium acetate, 10 [micro]M GDP, 1 mM dithiothreitol, 100 mM NaC1
, 5 mg bovine serum albumin per milliliter, 2.5 units (at 25[degrees]C) of adenosine deaminase per milliliter, and approximately 100,000 disintegrations per min (dpm) [[S.sup.35]]GTP[S] (0.3 to 0.5 nM).
chlorotica was homogenized in an ice-cold buffer (450 mM NaC1
, 1.0 mM EDTA, 5.0 mM 3-[N-morpholino]propane-sulfonic acid (MOPS), 2.3 [micro]M leupeptin, 1.0 mM dithiothreitol (DTT), 500 [micro]M phenylmethylsulfonyl fluoride (PMSF), pH 7.5) containing the mucolytic agent n-acetyl cysteine (500 mM), which is necessary to disperse the copious mucus produced by the slug (26).
To obtain final [Na.sup.+] concentrations of 200-400 mM, the 1/2 ASWs were supplemented with [Na.sup.+] in 50-mM increments using a stock solution of 4.4 M NaC1
. Fertilization assays in these media were conducted as described above.
Both swab heads were then vortexed for 30 sec in 5 mL of a sterile 3% NaC1
Also, rhizobial infection changed the levels of miR160, miR393, miR164, and miR168, which target ARFs (ARF10, ARF16, andARF17), TIR1, NAC1
, andAGO1, respectively.
The slides were then placed for 40 min in a freshly prepared lysis buffer (2.5 M NaC1
, 100 mM EDTA, 10 mM Tris, 1% Triton X-100, and 10% DMSO, pH 10).
Chua, "MicroRNA directs mRNA cleavage of the transcription factor NAC1
to downregulate auxin signals for Arabidopsis lateral root development," Plant Cell, vol.
* Second treatment: 50 meq of NaC1
; 2.9 [gl.sup.-1])
In 100mL Erlenmeyer titration flasks 40 mL of 0.10 M NaC1
solution was taken.