C Breast Tumors GST GSTPTEN AFYLDK ++ DBTRG LYSATE TPOSAPR P PTEN IP: P-REX2a IH
ID: 3167145 • Letter: C
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C Breast Tumors GST GSTPTEN AFYLDK ++ DBTRG LYSATE TPOSAPR P PTEN IP: P-REX2a IHNTAK SHGLPPR YLFRGR P-REX2a P-REX2 NKVPIIK MVP LLLELNK PTEN ?heavy chain PTEN Input O GFFSLTAH DEPLLFR PREX2a Input LVSTKPR LYKMMCR NKWFVCMAK YDDGTFYPR TYKSVVMANK P0.027 GFGPSVVHAVGR ?GR PTEN- PTEN+ 21 32 Fig. 1. P-REX2a as a PTEN binding protein. ? Silver stain of 1 M salt elu, enous PTEN and P-REX2a. Immun precipitations were performed (S) and pro- tions of affinity-purified PTEN-binding proteins. GST and GST-PTEN columns teins were detected by immunoblotting.(C) Box plot of P-REXZa expression in were incubated with (left and middle) and without (right) DBTRG-0SMG cyto PTEN-positive and -negative breast tumors. Bars above and below represent plasmic extract, and proteins eluted with high salt were separated and iden- the maximum and minimum expression, respectively. The box delineates the first to tified by mass spectrometry (9). P-REX2a and MVP are indicated with arrows third quartiles of expression, and the central bar represents the median. P-REX2a Peptide sequences of P-REX2a are shown. (B) Coimmunoprecipitation of endog levels are significantly associated with PTEN status (P-0.027) by two-tailed t testExplanation / Answer
1 -Silver staining is done when the very less protein is present in the lysate. In lane 1 only GST tagged plasmid is transfected to check if the cells lysate is bound to the GST-tagged bead.
GST-PTEN protein is immobilized on the bead and DBTRG-05MG lysate was passed (lane 2) and without the lysate (lane 3)
2- There are many bands in the lane in which lysate was passed. Out of which two proteins undergone mass spectroscopy to check the interacting partner of PTEN. MVP is a well known PTEN interacting protein which is a positive control for our binding experiment. Other protein is P-REX2a.
In order to check the interaction of PTEN and P-REX2a, cell lysate was prepared and Immunoprecipitation was dome with P-REX2a and probed with PTEN antibody. Western blot confirmed that P-REX2a interacts with PTEN endogenously in the cell. IP with PTEN and western blot with P-REX2a was also done to check the reverse interaction. The same result was obtained. Mouse IgG and Rabbit IgG were used to check the non-specific interaction.
Original lysate before adding the antibody for IP was used to check whether PTEN and P-REX2a were expressing in the cells or not.
3- In this figure, the expression levels of P-REX2a is compared between the cell lines which express PTEN and cell lines which don't and it was found that cell lines which PTEN also express high levels of P-REX2a as compared to cell line where PTEN-negative breast cancer cells
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