Fig 1: TCRP1 was highly expressed in many tumors and was positively correlated with expression of p-PDK1 and p-AKT. (a) Expression of TCRP1 was detected with PCR chip in a variety of tumors and corresponding normal tissue. (b) Expression of TCRP1, p-PDK1 and p-AKT1 were detected by immunohistochemistry in lung cancer and glioma. (c) Scatter plot showed relative abundance of TCRP1, p-PDK1 and p-AKT1 in lung cancer and glioma; *P <0.05 vs TCRP1. (d) The correlation trend of expression of TCRP1, p-PDK1 and p-AKT1 in glioma and lung cancer.
Fig 2: TCRP1 directly interacts with PDK1 and activates PDK1/AKT signaling.(a) The effects of TCRP1 on the expression of PI3K/Akt and its downstream molecules. (b) 293T and NIH/3T3 cells were co-transfected with EGFP-TCRP1 and pDsred-PDK1, or EGFP-TCRP1 and pDsred-AKT1, the direct interaction between TCRP1 and PDK1, or TCRP1 and AKT1, were determined by forster resonance energy transfer (FRET) assay. Cells co-transfected with EGFP-PDK1 plasmids, and pDsred-AKT1 was used as positive control. (c) Total protein extracts of NIH/3T3 cells were subjected to IP using TCRP1 antibody or control IgG, followed by western blotting (WB) with PDK1 antibody (upper panel). Reciprocal IP was done using PDK1 antibody or control IgG, followed by WB with the TCRP1 antibody (lower panel).
Fig 3: The PDK1-binding domain of TCRP1 is R93-S107 and T109-A124.(a) Motif analysis of TCRP1 was processed in Scansite database (http://scansite.mit.edu/), and showed that the 93–107 amino-acid and 109–124 amino-acid sequences in the TCRP1 were PDK1-binding motif (upper panel). Two vectors including the two TCRP1 mutant fragments were constructed named pEGFP-TCRP1/mut1 and pEGFP-TCRP1/mut2, respectively (lower panel). (b) NIH/3T3 cells were co-transfected with EGFP-TCRP1/mut1 and pDsred-PDK1, or EGFP-TCRP1/mut2 and pDsred-PDK1, the direct interaction between TCRP1/muts and PDK1 was determined by forster resonance energy transfer (FRET) assay. (c) NIH/3T3 cells were transfected with pEGFP-TCRP1/mut1, or pEGFP-TCRP1/mut2, total protein extracts were subjected to IP using TCRP1 antibody or control IgG, followed by WB with PDK1 antibody. Reciprocal IP was done using PDK1 antibody or control IgG, followed by WB with the TCRP1 antibody.
Fig 4: Inhibition of PDK1 reversed TCRP1-mediated cell transformation of NIH/3T3 cells. (a, b) NIH/3T3/TCRP1 cells or NIH/3T3/TCRP1 primary cells were treated with PDK1 inhibitor OSU-03012, or TCRP1 siRNA, the expression levels of p-PDK1, PDK1 and cyclin D1 were measured by western blotting (a); cell viability were measured by MTS assay (b). (c, d) NIH/3T3/TCRP1 cells or NIH/3T3/TCRP1 primary cells were treated with PDK1 inhibitor OSU-03012, clonogenic capacity were measured by plate cloning assay and soft agar cloning assay (c). Cells were fixed in ethanol, and stained with propidium iodide, and then DNA contents were determined by flow cytometry (d). The percentage of cells in each phase of the cell cycle (G1, S and G2/M) was indicated. Experiments were repeated three times. *P <0.05.
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