Fig 1.
p120 phosphorylation at Y228 is elevated in RCC samples.
Tissue microarrays (TMAs) of normal and tumor matched renal cell carcinoma (RCC) samples, stained by IHC for p120 using the pp120, pY228, 15D2 and F1aSH antibodies. The whole TMA stainings are shown in (A); a selected tumor-normal sample pair in (B); and details of the stainings of the selected pair in (C). Scale bars are shown on the bottom right in (B) and (C). (D) Quantitation of the signal of the TMA staining obtained by using pp120, pY228 and F1aSH; n = 15.
Fig 2.
p120 phosphorylation at Y228 is elevated in BC tissues.
(A) Breast normal and tumor (BC) samples stained by IHC for p120 using the pp120, pY228, and 15D2 antibodies. (B) Quantitation of the signal of the BC samples staining obtained by using pp120, pY228 and 15D2; n = 9.
Fig 3.
The pp120 antibody binds p120 around the T916 site and fails to recognize it, when it is phosphorylated.
(A) Schematic of the p120 protein, depicting the different domains, the phosphorylation sites (in red lines), the different p120 isoforms and the region used as the pp120 immunogen. (B) E-cadherin (Ecad), and pp120 western blots of the p120-mutated SW48 colon cancer cells transfected with the constructs shown (1–7) or without (8), lysed and immunoprecipitated with E-cadherin. (C) Western blot of HCT116 cell lysates using either 15D2, a monoclonal T916 (cl.1) and a polyclonal T916 antibody (pl.). Molecular weights are shown on the right. (D) Western blot of HCT116 cell lysates treated with or without λ phosphatase and blotted for the monoclonal T916 (cl.1) and 15D2. (E) p120-depleted A431 cells were transfected with either murine wild type p120 isoform 3 (mp120-3A) or a construct with the T916 site mutated to alanine (mp120-3A T916A), and blotted for the monoclonal T916 (cl.1) and 15D2 antibodies. (F) Westen blot of MDCK lysates using pp120, 15D2 or T916 (cl.1) after immunoprecipitation with the same antibodies.
Fig 4.
T916 phosphorylation is increased in RCC samples.
Normal (N) and tumor (T) matched RCC samples were lysed and subjected to western blot for pp120, T916, Y228, F1aSH. Actin is the loading control.
Fig 5.
Phosphorylation at Y228 but not at T916 is critical for the transforming ability of p120.
Breast cancer MDA-MB-231 cells were depleted of endogenous p120 and were transfected with: (A) vector control, full-length wild-type murine p120 isoform 1A (mp120-1A), or murine p120 isoform 1A where eight Src-targeted tyrosine phosphorylation sites, including Y228, were mutated to phenylalanine (mp120-8F); (B) vector control, murine wild type p120 (mp120-T916), or with murine p120 constructs where the T916 site was mutated to either alanine (mp120-T916A) or to glutamic acid (mp120-T916E). Cells in all cases were grown to soft agar and colonies were counted (mean ±SEM, n = 6; **p<0.01, *p<0.05, one-way ANOVA compared to vector control; n.s, non-significant). The western blots indicate expression of p120 using the 15D2 antibody in each case; Actin is the loading control.