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. 2016 Nov 10;473(22):4243–4254. doi: 10.1042/BCJ20160674

Figure 6. Staurosporine inhibits DMT1 transport and reduces serine phosphorylation.

Figure 6.

(A) Iron uptake by HEK293T(DMT1) cells was assayed after incubation with indicated concentrations of staurosporine in phosphate-buffered saline with glucose (PBSG)++ at 37°C for 20 min. The histogram represents three experiments done in triplicate and results shown are mean values ± SEM for inhibition, *P < 0.001. (B) Serine phosphorylation of DMT1 was determined by immunoprecipitation as described in Figure 3, except after a preincubation step with a phosphatase inhibitor in PBS++ at 37°C for 15 min, followed by assays containing a phosphatase inhibitor with 20 μM staurosporine or vehicle (0.5% DMSO, v/v) at 37°C for 20 min. Immunoprecipitation was performed using an anti-HA antibody (IP: HA) and immunoblotting was performed (IP: HA and IB: Phosphoserine) in comparison with immunoprecipitated DMT1-HA protein (IP: HA and IB: HA). Total cell lysates from the same samples (Input) were used to detect DMT1-HA protein (IB: HA) and actin was used as a loading control (IB: Actin). (C) Mean ratio of DMT1-HA: Actin ± SD from untreated cells (−) and cells treated with staurosporine (+) determined by densitometry of two independent experiments as described in B.