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. 2012 Apr 1;23(7):1376–1387. doi: 10.1091/mbc.E11-07-0634

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© 2012 Lee et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 4: — Netrin-2 induces phosphorylation of Stim1 at serine 575 via ERK activation. (A) The consensus ERK phosphorylation motif and a potential phosphorylation site at serine 575. (B) In vitro phosphorylation of the putative ERK phosphorylation site of Stim1 by ERK. The wild-type GST-Stim1 encompassing amino acids 542–607 or the alanine mutant form of GST-Stim1 at serine 575 (S575A) was used as substrate, and phosphorylation was measured by ³²P incorporation. GST serves as a negative control. The substrate levels were determined using fast green staining. p-GST-STIM1 and GST-STIM1 signals were quantified by densitometry. The signal ratio of GST-STIM1^WT was set to 1. (C) 293T cells transfected with Stim1 were treated with PD98059 for 1 h and analyzed by immunoblotting. Signals of p-NFATc3, NFATc3, p575-Stim1, Stim1, and β-tubulin were quantified by densitometry, and the ratio from control transfectants was set to 1. (D) Lysates of 293T cells cotransfected with control or Stim1 expression vector plus a scrambled siRNA or ERK2 siRNA were subjected to immunoblotting with antibodies to p-NFATc3, NFATc3, pS575-Stim1, Stim1, ERK2, and β-tubulin as a loading control. Signals of p-NFATc3, NFATc3, ERK2, and β-tubulin were quantified by densitometry; ratios are reported under each lane at the bottom in arbitrary units, with control transfectants set to 1. (E) Lysates from C2C12 cells treated with netrin-2 for indicated times were immunoblotted with antibodies to p-ERK1/2 or ERK1/2. (F) C2C12 cells were incubated for 8 h in DM and treated with netrin-2 for 1 h. For the treatment with PD98059, cells were pretreated with PD98059 for 1 h, prior to the netrin treatment. Lysates were immunoblotted with indicated antibodies. Signals of p-NFATc3, NFATc3, and β-tubulin were quantified by densitometry, and the ratio from the control transfectant was set to 1. (G) 293T cells transfected with Stim1 were treated with SB203580 for 1 h and analyzed by immunoblotting with antibodies to pS575-Stim1, Stim1, and β-tubulin as a loading control. Signals of pS575-Stim1 and Stim1 were quantified by densitometry, and the ratio from the control transfectant was set to 1.