Journal: Microbiology Spectrum

Article Title: Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery

doi: 10.1128/spectrum.03513-23

Figure Lengend Snippet: Subcellular colocalization of 24K and miRNA biogenesis proteins. ( A ) (i) Colocalization of 24K:mRFP with CFP:AtDCL1 in nuclear aggregates. ( B ) (i) Colocalization of 24K:mRFP with AtHYL1:YFP in nuclear aggregates. ( C ) (i) Colocalization of 24K:mRFP with AtSE:YFP in nuclear aggregates. (ii) The mRFP control does not colocalize in nuclear aggregates with any of the tested proteins. ( D ) Western blot analyses of extracts from N. benthamiana leaves in colocalization assays at 3 dpa. Anti-RFP (@RFP) or anti-GFP (@GFP) monoclonal antibodies were used for 24K or biogenesis proteins, respectively. Coomassie blue stain is shown as loading control. The numbers below correspond to normalized band density. Ratios between AtDCL and AtDCL1/24K (240 kDa); AtHYL1 and AtHYL1/24K (75 kDa); AtSE and AtSE/24K (110 kDa) are shown under the lines. Scale bar: 10 µm.

Article Snippet: GFP (or its variants, CFP or YFP) and RFP fusion proteins were detected with anti-GFP (3H9) monoclonal antibody (Chromotek, Germany) and anti-RFP (6G6) monoclonal antibody (Chromotek, Germany), respectively.

Techniques: Control, Western Blot, Bioprocessing, Staining

Journal: Microbiology Spectrum

Article Title: Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery

doi: 10.1128/spectrum.03513-23

Figure Lengend Snippet: Interaction between 24K protein and miRNA biogenesis proteins. ( A ) In vivo analysis of BiFC assays in epidermal cells of N. benthamiana plants. The merge panels show positive interaction between N-mCitrine:24K and C-mCitrine:AtHYL1 (i) and N-mCitrine:24K and C-mCitrine:AtSE (ii) and no interaction between N-mCitrine:24K with C-mCitrine:AtDCL1 (iii). (iv). Negative control: N-mCitrine:24K + C-mCitrine (empty vector). Chloroplasts were marked in blue in the case of the negative interactions. In all cases, Fib:mRFP was used as an expression control of infiltrated samples. Scale bar: 10 µm. ( B ) Co-IP assay between 24K:mRFP and AtHYL1. INPUT and UNBOUND controls correspond to input and output fractions without binding to bead system, respectively. The IP fraction corresponds to immunoprecipitated proteins. (−) corresponds to the negative control using beads without @HYL1 antibody. (+) corresponds to the immunoprecipitated samples with @HYL1 attached to the beads. The anti-RFP (@RFP) and anti-HYL1 (@HYL1) monoclonal antibodies were used in each case to develop the blots. The presence of both proteins in the IP fraction indicates a positive interaction. ( C ) Co-IP assay between the 24K:mRFP and AtSE:YFP protein. The INPUT and UNBOUND controls correspond to input and output fractions without binding to bead system, respectively. The IP fraction corresponds to immunoprecipitated proteins with @GFP attached to the beads. Free mRFP was used as a negative control. The anti-RFP (@RFP) and anti-GFP (@GFP) antibodies were used in each case to develop the blots. The presence of both proteins in IP fraction indicates a positive interaction.

Article Snippet: GFP (or its variants, CFP or YFP) and RFP fusion proteins were detected with anti-GFP (3H9) monoclonal antibody (Chromotek, Germany) and anti-RFP (6G6) monoclonal antibody (Chromotek, Germany), respectively.

Techniques: In Vivo, Negative Control, Plasmid Preparation, Expressing, Control, Co-Immunoprecipitation Assay, Binding Assay, Immunoprecipitation, Bioprocessing

Journal: Molecular Biology of the Cell

Article Title: Steady-state epithelial apical flatness is characterized by MLCK morphodynamics and asynchronous Ca 2+ oscillations, but not by underlying ECM geometry

doi: 10.1091/mbc.E25-12-0583

Figure Lengend Snippet: Association between apical flatness and the extracellular matrix (ECM). (A) Schematic of Rhodamine-laminin patches on Transwell membranes before MDCK cell culture. (B) Quantification of steep and shallow Rhodamine-laminin patches. The ratio of apical/basal area of the laminin patch delineated the two categories of steep and shallow patches. “Steep” patches had a ratio of 2.35 ± 0.26 s.d., and “shallow” patches had a ratio of 1.74 ± 0.31 s.d. Note: Both steep/shallow patches were clearly delineated by a ratio that was either > or < 2, with the ‘steep’ patch ratio > 2, and “shallow” patch ratio < 2 ( n = 16 laminin patches). (C, C’) Two types of epithelial response in morphology are based on the steepness of the laminin patch. Cells with complete, partial, or little to no basement membrane positioned on the patch were defined as center, peripheral, or outside, respectively. (D) Top and side views of stacked confocal optical sections of a Type 1 MDCK confluent monolayer immunostained with β-catenin (green), filamentous actin dye, phallodin (grey), and a DNA DAPI (blue) counterstain when grown on a “steep” Rhodamine-laminin patch (red). (E) As in (D) but of a Type 2 MDCK confluent monolayer growing on a “shallow” Rhodamine-laminin patch (red). (F) Macro-height measurements for Type 1 (grey) and Type 2 (blue) monolayers. Individual cell height analysis was performed based on the relative position on the laminin patch. Average macro-height of center, peripheral, or outside cells was 8.10 µm, 8.42 µm, and 8.64, respectively ( n = 149 cells). For Type 2 cells, the average macro-height for center, peripheral, and outside cells was 4.60 µm s.d., 5.47 µm, and 8.12 µm, respectively ( n = 117 cells). (G) As in (F), but of micro-height measurements. For Type 1 cells, average micro-height measurements for center, peripheral, and outside cells were 1.29 µm, 1.36 µm, and 1.37 µm, respectively ( n = 149 cells). For Type 2 cells, the average micro-height for the center, peripheral, and outside cells was 1.12 µm, 1.16 µm, and 1.15 µm, respectively ( n = 117 cells). (H) Standard deviations of macro- and micro-height of Types 1 (steep) and 2 (shallow) MDCK confluent monolayers. Flatness threshold indices established in are indicated with horizontal dashed lines. Note: Type 1 epithelium loses flatness, while Type 2 epithelium maintains flatness by modulating individual cellular behaviors on the laminin patch. (Scale bars: 8 µm, 20 µm, 7 µm).

Article Snippet: Rhodamine-laminin (#LMN01-A, Cytoskeleton) was applied to Transwell membranes.

Techniques: Cell Culture, Membrane