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. 2022 Aug 9;14(16):3847. doi: 10.3390/cancers14163847

Figure 2.

Figure 2

Sam68 interacts with ATM kinase, and it is phosphorylated on SQ/TQ motif upon DDR induction. (A) HEK293T cells were transiently transfected with a combination of different constructs that allow the overexpression of the fusion proteins GFP-Sam68 (pCDNA GFP-Sam68) and ATM-Flag (pCDNA FLAG-ATM) as indicated. Representative western blot of total protein extracts (whole-cell lysate, WCL) (293T WCL) and protein extracts subjected to co-immunoprecipitation assay. Co-immunoprecipitations were performed using anti-GFP (IP: GFP) or anti-FLAG (IP: FLAG) antibodies and immunoblotted with the indicated antibodies. (B) LNCaP cells were treated, or not, with KU55933 10 μM for 2 h and/or neocarzinostatin (NCS) 500 ng/mL for 1 h. Representative western blot of total protein extracts and protein extracts (LNCaP WCL) subjected to co-immunoprecipitation assay using anti-ATM (IP: ATM) antibody and IgG (IgG) as negative control. (C) HEK293T cells were transiently transfected as in (A) and treated, or not, KU55933 10 μM for 2 h. Representative western blot of WCL (293T WCL) and protein extracts subjected to immunoprecipitation assay using anti-GFP (IP: GFP) and immunoblotted with the indicated antibodies. Bar graph shows the densitometric analysis of pSQ/TQ signals with respect to GFP (western blot assays) in IP experiments (n = 3; mean ± s.d., *** p < 0.001, Student’s t test). (D) PC LNCaP cells were treated, or not, with KU55933 10 μM for 2 h and/or neocarzinostatin (NCS) 500 ng/mL for 1 h. Representative western blot of total protein extracts and proteins co-immunoprecipitated with anti-Sam68 antibody (IP: Sam68).