Journal: PLoS ONE

Article Title: Time-Dependent Decay of mRNA and Ribosomal RNA during Platelet Aging and Its Correlation with Translation Activity

doi: 10.1371/journal.pone.0148064

Figure Lengend Snippet: On day 8, washed PLTs from DT- or saline-treated mice were resuspended in Tyrode’s albumin/DMEM medium and incubated at 37°C for different periods of time. (A) PLTs were incubated for 0, 2, 4, 6 and 24 h. The percentage of TO bright PLTs, determined at 0, 6 and 24 h, was 82, 33 and 2%, respectively. At each time points, PLT samples were fixed and permeabilized, stained with the anti-rRNA mAb Y10b and A647-conjugated anti-mouse IgGs, counterstained with an A488-conjugated anti-GP1bβ mAb and analyzed by confocal microscopy. PLT outlines, determined by Photoshop analysis of anti-GP1bβ staining, are depicted. Representative micrographs of analyzed cells from DT-treated animals (+DT) at times 0, 4 and 24 h and from saline-treated animals (-DT) at time 0 are shown. (B) The mean fluorescence intensity per unit area in each PLT stained with Y10b (+) or an isotype-matched control antibody (C) (y-axis, log2 scale) was calculated as described in the methods; 500 to 550 PLTs from DT-treated mice (+) or saline-treated mice (-) were analyzed for each condition. A one way ANOVA analysis followed by a Bonferroni post-hoc test comparing each pair of conditions gave p values of <0.001 (continuous bars) or <0.01 (dashed bars). Other combinations of Y10b stained samples were not statistically different.

Article Snippet: The anti-rRNA mAb Y10b (5 μg/mL, mouse IgG3, Novus Biologicals) was used to label ribosomes and revealed with A647-conjugated goat anti-mouse IgG.

Techniques: Saline, Incubation, Staining, Confocal Microscopy, Fluorescence, Control

Journal: The Journal of Cell Biology

Article Title: Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids

doi: 10.1083/jcb.202004079

Figure Lengend Snippet: Cytological evaluation and biochemical assay of ribosomal RNA binding by S9.6. (A) Representative image of HeLa cells treated with 100 µM PladB for 4 h to induce the formation of splicing speckles. Splicing speckles were revealed using an antibody targeting SF3B4, a component of the U2 spliceosome (red). Cells were costained with S9.6 (green). The inset highlights a representative cell with clearly demarcated SF3B4 and S9.6 signals. (B) Representative image of HeLa cells stained with the anti-rRNA antibody Y10b (green, left panel). Cells were costained with an engineered rabbit S9.6 antibody (red, middle panel). A merged image with DAPI staining is shown at right. Scale bar, 10 µm for A and B. (C) Quantification of whole cell mean Y10b intensities for individual cells that were mock- or enzyme-treated. Boxplots represent combined data from two biological replicates ( n indicates the number of cells quantified). (D) Native gel shift assays using purified human 40S (left) and 60S (right) ribosomes. Purified ribosomes (100 nM) were incubated for 10 min with increasing concentrations of S9.6 and Y10b, subjected to gel electrophoresis, and visualized with ethidium bromide staining. Red lines mark the approximated center of each band.

Article Snippet: When indicated, an engineered rabbit S9.6 antibody (Ab01137-23; Absolute Antibody) and/or the mouse anti 5.8S rRNA Y10b antibody (NB100-662; Novus Biologicals) was used for staining at 1:1,000 dilutions.

Techniques: RNA Binding Assay, Staining, Gel Shift, Purification, Incubation, Nucleic Acid Electrophoresis

Journal: The Journal of Cell Biology

Article Title: Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids

doi: 10.1083/jcb.202004079

Figure Lengend Snippet: Assessment of Y10b RNA:DNA hybrid and RNA binding in vitro and in situ. (A) Left: 54-mer RNA:DNA hybrid oligonucleotide substrates (100 nM) were incubated with increasing concentrations of S9.6 and Y10b, and binding was evaluated after gel electrophoresis and ethidium bromide staining. Right: Quantification of gel shift assays comparing the affinity of Y10b for RNA:DNA hybrids to that of S9.6. (B) Representative images of methanol-fixed HeLa cells that were mock-treated or pretreated with RNase T1 and RNase H for 1 h at room temperature before staining with the Y10b antibody. (C) Representative images of methanol-fixed HeLa cells stained with mouse S9.6 (green) and an engineered rabbit S9.6 (red). Scale bar, 10 µm.

Article Snippet: When indicated, an engineered rabbit S9.6 antibody (Ab01137-23; Absolute Antibody) and/or the mouse anti 5.8S rRNA Y10b antibody (NB100-662; Novus Biologicals) was used for staining at 1:1,000 dilutions.

Techniques: RNA Binding Assay, In Vitro, In Situ, Incubation, Binding Assay, Nucleic Acid Electrophoresis, Staining, Gel Shift