Fig. 1: Kindlin-2 acts as a scaffold to promote the AR-Src association and facilities Src-mediated AR Tyr-534 phosphorylation. | Cell Death & Disease

Fig. 1: Kindlin-2 acts as a scaffold to promote the AR-Src association and facilities Src-mediated AR Tyr-534 phosphorylation.

From: Kindlin-2 promotes Src-mediated tyrosine phosphorylation of androgen receptor and contributes to breast cancer progression

Fig. 1

A Volcano plots of the enriched native tyrosine phosphorylation proteins upon EGF stimulation in BT549 cells transfected with control siRNA (Ctrl siRNA) or Kindlin-2 siRNA (K2 siRNA) using Src superbinder Photo-pTyr-scaffold approach [86]. Red dots indicated the significantly enriched AR and Kindlin-2 (FERMT2). A complete list of the enriched proteins in control vs. Kindlin-2 knockdown BT549 cells was shown in Supplementary Table S1. False discovery rate (FDR) = 0.05, s0 = 2, two-sample t-test, n = 2. B Immunoblotting analysis of total AR and AR Tyr-534 phosphorylation level in control (Ctrl siRNA) and Kindlin-2 knockdown (K2 siRNA) BT549 cells or MDA-MB-453 cells with or without EGF stimulation (upper panel). Quantification data were shown in the lower panel. *p < 0.05 vs. Ctrl siRNA, n = 6 for BT549 cells; n = 4 for MDA-MB-453 cells. C BT549 (left panel) or MDA-MB-453 (right panel) cell lysates were immunoprecipitated with anti-Kindlin-2 antibody or mouse control IgG (mIgG) followed by immunoblotting with antibodies as indicated. The presence of Kindlin-2, AR and Src in cell lysates was shown as input. D BT549 (left panel) or MDA-MB-453 (right panel) cell lysates were immunoprecipitated with anti-AR antibody or rabbit control IgG (rIgG) followed by immunoblotting with antibodies as indicated. The presence of Kindlin-2, AR and Src in cell lysates was shown as input. E Control (Ctrl siRNA) and Kindlin-2 knockdown (K2 siRNA) BT549 cells lysates were immunoprecipitated with anti-AR antibody followed by immunoblotting with antibodies as indicated. The presence of Kindlin-2, AR and Src in cell lysates was shown as input. F Mapping the subdomains of Kindlin-2 that mediated the association with AR. Upper panel: schematic illustration of various Kindlin-2 fragments that were used in the GST pull-down assay. Lower panel: GST-fusion proteins containing various fragments of Kindlin-2 were used to pull-down endogenous AR from BT549 cell lysates. K2 Kindlin-2, AR androgen receptor.

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