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Figure 3 | Proteome Science

Figure 3

From: Automated quantitative multiplex immunofluorescence in situ imaging identifies phospho-S6 and phospho-PRAS40 as predictive protein biomarkers for prostate cancer lethality

Figure 3

Validation of PTEN, CCND1, SMAD4, SPP1, pS6 and pPRAS40 antibody specificity. Doxycycline-inducible shRNA knockdown cell lines were established for PTEN (A), CCND1 (B) and SMAD4 (C). Doxycycline treatment reduced the abundance of the target protein in all cases as assessed by WB. Cell lines with high or low/negative levels of expression of PTEN (A), CCND1 (B) and SMAD4 (C) were also examined by WB and IHC to further validate the specificity of the antibodies. SPP1 (D) antibody detected an SPP1-specific band and an additional band at a lower molecular weight as assessed by WB in PC3 cells, while the SPP1-specific upper band was significantly decreased in low SPP1-expressing BxPC3 cells. The staining intensity of the SPP1 antibody in PC3 and BxPC3 cells by IHC correlated well with the relative intensity of the SPP1-specific band detected by WB. The specificity of pS6 and pPRAS40 antibodies (E) was validated in DU145 cells. LY294002 treatment significantly reduced phosphorylation of RPS6 and AKT1S1 (PRAS40), as shown by WB and IHC, respectively.

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Proteome Science

ISSN: 1477-5956