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To this end we used a tester construct that included genomic SUB coding sequence (see below).
Therefore, reduced proteasomal activity should lead to incomplete proteolysis of a cODC1-containing tester construct.
Using the cyan fluorescent protein (CFP) containing tester construct CFP-cODC1-TDegF-RFP, we observed that pTEV + protease expression led to accumulation of cleavage intermediates in pre1-1 pre1-1 or pre1-1 pre4-1 mutant cells [ 39, 40] indicating incomplete proteolysis of CFP-cODC1-TDeg and F-RFP fragments.
Claw testing was performed over the typical site of sole ulcer (axial sole-bulb junction) and the central part of the dorso-abaxial claw wall of the front limbs by applying a large standard hoof-tester constructed with a force measuring device (KERN FK1K, Kern Kraft & Sohn, Balingen, Germany) welded on to one of the arms.
In addition, we performed flow cytometry measurements of cells expressing tester constructs (see Additional file 1 Figure S1A, B).
In order to simulate these conditions a reversed axisymmetrical material tester is designed and constructed.
We already constructed a tester plasmid pCETD-12 [ 22].
Both forward (apical cell/basal cell, apical cell cDNA as tester) and reverse (basal cell/apical cell, basal cell cDNA as tester) subtracted cDNA libraries were constructed to enrich the genes specifically or predominantly expressed in the apical or basal cells.
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