L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

25 avril 19411941/04/25 (A41,N14694)

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

Antibodies tested in this study.

<p>Antibodies tested in this study.</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

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

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

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