Antibodies that detect SHB in western blot fail to immunoprecipitate it.

<p>HEK 293T CE12 cells having one of their alleles tagged with V5 (V5-tag) or HEK 293T cells knock-out for Shb (KO) were used to prepare whole cell extracts, which were then used as material for the immunoprecipitations (40 μg of total protein). Shb was immunoprecipitated from the cell extracts (2 mg of total protein) using the 74483 and 530 antibodies from Santa Cruz. An anti-V5 mouse antibody was used for control immunoprecipitations. The samples were then analysed by western blot using a rabbit anti-V5 antibody. The arrowhead indicates the band corresponding to Shb. The bottom panel shows a lower exposure of the indicated area to better distinguish the heavy chain of the antibody used in the immunoprecipitation (indicated by *), against which the goat anti-rabbit secondary antibodies react (weakly in the case of mouse primary antibodies).</p

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Shb is located in the cytoplasm and in the nucleus.

<p>HEK 293T cells, treated with either control or Shb siRNA, were used to isolate nuclear and cytoplasmic cell fractions. These were analysed by western blot using a cytoplasmic marker (α-tubulin), a nuclear marker (PARP) and Shb.</p

Endogenous Shb-V5 can be detected simultaneously by an anti-V5 antibody and a Shb antibody.

<p>CE12 cells, which have one of their Shb allele tagged with V5, were treated with control or Shb-directed siRNAs. Western blots of the cell extracts were performed using simultaneously a Shb-specific antibody (panel A: Santa Cruz 530; panel B: Santa Cruz 74483) and an anti-V5 antibody. The Santa Cruz 530 antibody detected the same band as the anti-V5 antibody (arrowhead) but also many non-specific bands (filled stars), including an intense one in the proximity of the Shb band itself (open star). The Santa Cruz 74483 antibody detected the same band as the anti-V5 antibody (arrowhead) with much more affinity than the closest non-specific band (open star).</p

Assessment of nine anti-Shb antibodies.

<p>Anti-Shb antibodies were tested by western blotting for their ability to recognize their antigen in HEK 293T cells transfected with either empty plasmid or a Stag-Shb-V5 plasmid, or in CE12 cells, treated with control or Shb-specific siRNA. Whole cell lysates are those that were used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188311#pone.0188311.g001" target="_blank">Fig 1</a>. Arrowheads point to Shb-V5; *, unspecific signals.</p

Antibodies tested in this study.

<p>Antibodies tested in this study.</p

V5-tagged Shb is specifically detected with an anti-V5 antibody.

<p>The V5 signal corresponding to Shb-V5 was detected by western blot. HEK 293T cells were transfected with empty plasmid or a Stag-Shb-V5 encoding plasmid. To test the specificity of the signals, the CE12 cell line bearing an endogenous V5-tagged Shb allele was knocked-down for Shb (siRNA^Shb) or treated with control siRNA (siRNA^SCR). Arrowheads point to Shb-V5. Equal amounts of proteins were loaded (50 μg) on each lane.</p

Fragment N does not regulate survivin expression.

<p><b>A.</b> MIN6 cells were co-transfected with a luciferase expressing vector under the control of either a minimal promoter sequence allowing the transcription of survivin (left graph) or the entire survivin promoter sequence (right graph) with increasing amounts of fragment N- (closed circles) or E2F1- (open circles) encoding plasmids. The results correspond to the mean ±95% CI of 6 (left panel) and 3 (right panel) independent experiments performed in triplicate. Repeated measures ANOVA tests were performed to determine if there was a significant increase in luciferase activity induced by the E2F1- or fragment N-encoding plasmids (normality was verified with the Shapiro-Wilk test). <b>B–C</b>. MEFs were infected with an empty virus or with a lentivirus encoding the HA-tagged version of the D157A fragment N mutant. Survivin mRNA levels were analyzed 24 hours later by quantitative RT-PCR (panel B). The location of the 672FW and 672RV primers (red arrows), used for the amplification of the survivin mRNA, is depicted on top of the graph. Alternatively, cells were lysed and the protein expression of HA-fragment N and survivin was assessed by Western blotting (panel C). The results correspond to the mean ±95% CI of 3 independent experiments. <b>D.</b> Skins of mice were irradiated with low (0.05 J/cm²) and high (0.3 J/cm²) doses of UV-B light. Expression of nuclear and cytoplasmic survivin was assessed by immunofluorescence <i>in situ</i> (left panel). The quantitation shown on the right-hand side corresponds to percentages of keratinocytes displaying nuclear or cytoplasmic survivin (mean ±95% CI of 6 and 10 mice for the low and high UV-B dose exposure, respectively). No cells were found to display both cytoplasmic and nuclear survivin expression.</p

Fragment N does not affect cell cycling.

<p>HeLa cells, infected with an empty virus or with a lentivirus encoding the HA-tagged version of the D157A fragment N mutant, were synchronized at G1 by treatment with mimosine for 18 hours. The cells were then washed and cultured in fresh medium for the indicated periods of time. <b>A.</b> Representative histograms obtained at different time points after release from the mimosine block (NT, not synchronized cells). <b>B.</b> Immunocytochemistry-based detection (gold staining) of fragment N in the infected cells. The nuclei are colored in blue (Hoechst staining). <b>C.</b> Percentage of cells in each phase of the cell cycle as determined by PI staining (DNA content). Results represent the mean ±95% CI of 4 independent experiments.</p