Typhoon fla 9000

Blots from Figs 1–6 were obtained by autoradiography using GE Healthcare Amersham Hyperfilm^TM ECL. Western blots were scanned using Epson Perfection 1670 Scanner and quantified using an image processor software (ImageJ). Blots from Fig. 7 and Supplementary Figs 4–5 were obtained using the Odyssey Infrared Imaging System (LI-COR Biosciences). BRET experiments were performed using the Mithras LB 940 (Berthold technologies). Uptiblue measurements were performed using a Typhoon^TM FLA 9000 (GE Healthcare).

After ⁶⁵Zn administration, plants were harvested and roots were blotted dry and laid out on an absorbent pad for imaging. Shoots were also laid out on a separate absorbent pad for imaging. Radiographic images of different tissue areas (roots and shoots) were acquired by exposing phosphor plate films. Phosphor plates of roots were exposed for 36 h while plates of shoots were exposed for 120 h to acquire a sufficient signal. After exposure, phosphor plates were then read using the Typhoon 9000 imager (Typhoon^TM FLA 9000, GE Healthcare, Piscataway, NJ, USA). Images were only used qualitatively for determining spatial patterning of ⁶⁵Zn tracer in roots and shoots; hence no attempt was made to normalize image data. Comparative whole-plant radiographic images of ⁵⁹Fe³⁺ and ⁵⁹Fe²⁺ were also acquired from our prior work [16 (link)], but because of the faster decay rate of this radionuclide (t_½ 44.5 day), we only needed to expose these tissues for 16 h.

Subsequent to ¹¹C exposures, plants were harvested, roots were laid out, and radiographic images acquired by exposing phosphor plate films for on average 10 min. Phosphor plates were read using a Typhoon 9000 imager (Typhoon^TM FLA 9000, GE Healthcare, Piscataway, NJ, USA). Images were used qualitatively only for determining spatial patterning of ¹¹C activity (Figure 2).

Total RNA was isolated using the SV Total RNA Isolation System (Promega) as described [52 (link)]. 5’ RACE was performed using the 5’/3’ RACE Kit (Roche). For Northern blotting 10–20 μg total RNA were separated using 7 M urea/10% acrylamide/0.6 x TBE gels. The RNA was transferred onto positively charged membranes (Roche) by semi dry blotting in 1 x TBE and UV crosslinked. For radioactive labeling 20 μCi γ-ATP³² (Hartmann Analytic) were mixed with 10 U T4 Polynucleotide kinase (Fermentas) and 4 pmol oligonucleotide and incubated at 37°C for 1 h. The reaction was stopped by addition of STE buffer (100 mM NaCl, 10 mM Tris, pH 8, 1 mM EDTA) and the labeled oligonucleotides were purified using MicroSpin G-25 columns (GE Healthcare). The probes were mixed with 500 μg yeast tRNA (Invitrogen) and 250 μg salmon sperm DNA (Invitrogen) to reduce unspecific probe binding. Denaturation was carried out for 10 min at 95°C. Hybridization was performed in hybridization buffer (0.5 M Na₂HPO₄, pH 7.2, 1 mM EDTA pH 7.5, 7% [w/v] SDS) over night at 68°C. The blots were washed in washing buffer (40 mM Na₂HPO₄, pH 7.2, 1 mM EDTA, pH 7.5, 1% [w/v] SDS) and exposed to a Phosphorimaging screen and analyzed with a Typhoon FLA-9000 (GE Healthcare).

ExoG (1.25, 25 or 50 nM) was incubated with FAM (6-carboxyfluorescein)-labeled substrates (100 nM) in a 10 μL reaction containing 100 μg mL⁻¹ bovine serum albumin (BSA), 10 mM HEPES pH 7.4, 150 mM NaCl and 2.5 mM MgCl₂ at 37°C. For biotin-labeled substrates, an additional 200 nM NeutrAvidin (Thermo Scientific) was included in the reaction. Oligonucleotide sequences are listed in Supplementary Table S1. Reactions were stopped at the indicated time-points by adding an equal amount of 2X TBE/urea sample buffer (BIO-RAD) and heating at 65°C for 20 min. To fully release the FAM-labeled probe from the complementary strand, 2 μL of 100 μM competitive DNA oligonucleotides was added. The resultant mixtures were heated at 95°C for 5 min, then at 30°C for 10 min, before being gradually cooled to room temperature (∼20°C). The solutions were loaded and separated by a 20% denatured acrylamide gel containing 6 M urea. FAM-labeled oligonucleotides (excitation at 473 nM and emission at 520 nM) in the resultant gels were visualized using a Typhoon FLA 9000 biomolecular imager (GE Healthcare Life Sciences). Quantification of band signal was plotted in GraphPad Prism v. 7.0 (34 ).

Retinal protein extracts were prepared as previously described [31 (link)]. Briefly, samples (20 μg of protein) were separated in 10%, 12%, or 15% sodium dodecyl sulphate-poly(acrylamide) gel electrophoresis (SDS-PAGE), and the proteins were transferred to polyvinylidene difluoride membrane (Millipore, Madrid, Spain). The membranes were blocked in 5% skim milk in Tris-buffered saline (TBS: 137 mM NaCl, 20 mM Tris-HCl, pH 7.6) containing 0.1% Tween-20 (TBS-T) for 1 h at RT. Afterwards, membranes were incubated with the primary antibodies (Supplementary Table S1), followed by incubation with respective alkaline phosphatase-conjugated secondary antibody for 1 h at RT. Protein detection was conducted using ECF™ (GE Healthcare Amersham™, Chalfont Saint Giles, UK) in accordance with the manufacturer’s instructions on Typhoon FLA 9000 (GE Healthcare Bioscience AB, Uppsala, Sweden). Immunoblots were reprobed with glyceraldehyde 3-phosphate dehydrogenase antibody (GAPDH, 1:5000; Life Technologies, Cambridge, UK) to ensure equal sample loading. Digital quantification of band intensity was performed using Image Studio 5.2 software (LI-COR Biosciences, Lincoln, NE, USA).