Journal: Eukaryotic Cell

Article Title: Copper-Dependent Iron Assimilation Pathway in the Model Photosynthetic Eukaryote Chlamydomonas reinhardtii

doi: 10.1128/EC.1.5.736-757.2002

Figure Lengend Snippet: Fox1 abundance in iron-deficient Chlamydomonas . Protein extracts from C. reinhardtii cells grown in TAP medium containing various concentrations of copper and iron, and collected at a density of 10 7 cells ml −1 , were prepared as described in Materials and Methods. Extracts were analyzed after separation by denaturing gel electrophoresis (7.5% acrylamide), transfer to nitrocellulose (1 h, 100 V, in 25 mM Tris-192 mM glycine-0.04% [wt/vol] SDS-20% [vol/vol] methanol), and incubation with anti-Fox1 antiserum (1:300 dilution). Bound antibody was visualized colorimetrically after incubation with alkaline phosphatase-conjugated secondary antibody. (A) The Fox1 gene product accumulates in −Fe cells. Cells grown in medium containing 200 μM added Fe-EDTA were transferred to fresh medium supplemented with 0.1, 0.25, 1, 18, or 200 μM Fe-EDTA and sampled after 5 days of growth. Protein extracts from 2.5 × 10 6 cells were analyzed as described above. Samples were normalized for loading on the basis of equal cell numbers and verified for accumulation of CF 1 , which is iron independent (see Materials and Methods). Percentages on the left are the fractional amounts of the “0.1 μM Fe” sample that were loaded. (B) Time course of Fox1 induction in −Fe cells. Cells grown with 200 μM added Fe-EDTA were transferred to fresh medium lacking iron (−Fe) or containing 200 μM added Fe-EDTA (+Fe). Cultures were sampled each day for 5 days (d), and ferroxidase abundance was analyzed as described above. Percentages shown on the right arethe amounts of the day 1 sample that were loaded. The accumulation of the α and β subunits of CF 1 is shown as a loading control. (C) Time course of Fox1 mRNA accumulation upon transfer from Fe-replete (200 μM) to Fe-deficent (0 μM) medium. Cultures were sampled at the indicated time after transfer for RNA isolations. The RNAs were analyzed by blot hybridization. The Cβlp mRNA was used as an internal control. Its behavior as a function of cell growth and iron nutrition is typical of many other RNAs. (D) Cu is required for accumulation of Fox1. C. reinhardtii was cultured in copper-supplemented (6 μM, +Cu) or copper-deficient (no added copper, −Cu), iron-supplemented (18 μM, +Fe) or iron-deficient (no added iron, −Fe) TAP medium. One microliter of protein extract from 4 × 10 8 cells was analyzed as described above. Percentages on the left are the fraction of the −Fe-+Cu sample that was applied for quantitation. Molecular masses of markers (Gibco BRL) are indicated in kilodaltons. (E) Cu-dependent accumulation of Fox1 in iron-replete cells. Copper-deficient, iron-replete (18 μM) CC125 cells were transferred to fresh (0 μM supplemental iron) medium containing the indicated amounts of copper and sampled for immunoblot analysis of Fox1 accumulation after 3 days. Each lane was loaded with material from 2 × 10 7 cells (equivalent to approximately 5 μg of chlorophyll). Percentages on the right are the fraction of the sample from medium containing 100 μM supplemental copper.

Article Snippet: Cultures were chilled in ice-water; collected by centrifugation (4,300 × g , 15 min); washed once with a solution of cold 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0), and 100 mM NaCl (TEN); resuspended in 10 ml of cold 50 mM Tris HCl (pH 7.5) containing 5 mM EDTA; subjected to three quick-freeze (dry ice ethanol)-quick-thaw (37°C) cycles; and disrupted by sonication (Fisher Scientific model 550 Sonic Dismembrator; microtip probe, amplitude setting 4, 12 cycles of 30 s of sonication followed by 60 s of cooling).

Techniques: Nucleic Acid Electrophoresis, Incubation, Hybridization, Cell Culture, Quantitation Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Efficiency of Incorporation and Chain Termination Determines the Inhibition Potency of 2′-Modified Nucleotide Analogs against Hepatitis C Virus Polymerase

doi: 10.1128/AAC.02666-14

Figure Lengend Snippet: Chain termination in the presence of the next correct nucleotide. High-resolution Tris-borate-EDTA–urea gel electrophoresis showing the extension of 10- to 11-mer RNA products with UTP or UTP analogs, followed by the addition of the next correct

Article Snippet: Solutions of MgCl2 , EDTA, NaCl, and Tris-Cl buffers were purchased from Ambion (Austin, TX).

Techniques: Nucleic Acid Electrophoresis

Journal: The Journal of Cell Biology

Article Title: Tetrahymena thermophila contains a conventional ?-tubulin that is differentially required for the maintenance of different microtubule-organizing centers

doi: 10.1083/jcb.200205101

Figure Lengend Snippet: α -Tubulin localization in progeny of knockout heterokaryons. GTU1 -KO heterokaryons were mated in 10 mM Tris for 24 h, refed with SPP, and fixed at different times (given in hours) after refeeding. (A) Immunofluorescence staining of α-tubulin (DM1A; a–e) and of DNA (DAPI; f–i) in knockout heterokaryon progeny cells. (a) No visible OA with normal cell shape and CVPs. (b) No visible OA and CVPs; with visible longitudinal cortical MT structure (LG-MT). (c) Round cells without visible OA and CVPs. (d and e) No visible OA and CVPs with misplaced division furrow. (f–i) Cells with abnormal nuclear phenotypes. (f) Two dividing Mics (same cell as c). (g) Elongated dividing Mics. (h) Abnormal Mac (same cell as d). (i) Degrading Macs. (B) Summary of appearance of the five categories of cells after cadmium depletion. The percentage of cells without OA or with abnormal OP development (ΔOA) was first determined. These ΔOA cells were then classified into four categories as shown in Fig. 4 B and the percentage of ΔOA cells in each category was calculated.

Article Snippet: Western blot analysis To demonstrate expression of HA-tagged γ-tubulin, CU428, tG2N-HA, and cTTMG-HA strains were grown to 2 × 105 cells/ml in SPP/CdCl2 (1.0 μg/ml) at 30°C, harvested and washed in 10 mM Tris-Cl, pH 7.5, containing protease inhibitors (Complete Mini; Roche).

Techniques: Knock-Out, Immunofluorescence, Staining, Magnetic Cell Separation