The Biochemical Properties of the Arabidopsis Ecto-Nucleoside Triphosphate Diphosphohydrolase AtAPY1 Contradict a Direct Role in Purinergic Signaling
Fig 4
Purification of AtAPY1 and AtAPY1-δTM.
The explanation of the colors in the schematic representations of AtAPY1 and AtAPY1-δTM can be found in Fig. 2. (A) Total proteins from 1.6 x 105 HEK293 cells were harvested at each of the indicated time points post transfection with AtAPY1 DNA, separated in a 4–12% gradient gel under denaturing conditions, transferred onto a PVDF membrane and successively incubated with anti-APY1 and anti-His antibodies (left panel). The black arrows mark the signal specific for AtAPY1, while the gray arrowheads indicate unspecific bands. The right panel shows the total protein extract from 1.4 x 108 HEK293 cells harvested at 89 h after transfection with AtAPY1 DNA subjected to Ni2+-affinity chromatography. Various fractions were separated in a 4–12% gel under denaturing conditions and either stained with Coomassie or transferred onto a PVDF membrane for Western blot analysis. The black arrow indicates the signal detected with antibodies against AtAPY1. The volumes loaded were 1/480 of the flow through (FT) fraction, 1/50 of each of the final two wash fractions W3 and W4 and 1/100 of the elution fraction E. (B) The left panel shows samples representing equal volumes (1/3,000) of the culture medium of 1 x 108 HEK293 cells taken at the indicated time points post transfection with AtAPY1-δTM DNA and separated in a 4–12% gradient gel under denaturing conditions. Subsequently, the proteins were either stained with Coomassie or blotted onto a PVDF membrane for Western blot analysis. The right panel depicts the culture medium of 4 x 107 HEK293 cells at time point 88 h after transfection with AtAPY1-δTM DNA subjected to Ni2+-affinity chromatography. A gradient gel (4–12%) was loaded with 20 μL of supernatant (S) and 20 μL of flow through (FT), 10 μL of each wash 1–5 and 10 μL of each eluate 1–2. For total volumes of the individual fractions see Materials and Methods. The protein amount loaded for eluate 1 equals about 70 ng. Following SDS-PAGE, the gel was silver-stained.