Fig. 1

Maspin nuclear translocation depends on EGFR. A Starved MCF-10A cells were treated with EGF (20 ng/mL) for the indicated periods of time. Nuclear and cytoplasmic fractions were prepared and maspin protein levels were analyzed by immunoblot. Fractionation efficiency was monitored by reprobing the membrane with anti-lamin A/C and anti-α-tubulin. B Starved MCF-10A cells were left untreated (−) or treated with 20 ng/ml EGF for 1 h (+). Whole cell lysates were immunoprecipitated (IP) with anti-maspin or anti-EGFR, as indicated. Isotype matched IgG was used as a negative control. EGFR and maspin co-immunoprecipitation and input samples were evaluated by immunoblot (IB), as indicated on the right side of the figure. C Starved MCF-10A cells were pretreated with EGFR inhibitor gefitinib or vehicle (DMSO) for 30 min followed by 20 ng/ml EGF for additional 1 h. Maspin localization was analyzed by immunofluorescence with anti-maspin antibody. D MCF-10A cells were transiently transfected with Stealth Select RNAi negative control or two different siRNA against EGFR. EGFR silencing was evaluated by immunoblot with anti-EGFR. α-Tubulin was probed as a loading control. E 48 h after transfection cells were treated with 20 ng/ml EGF for 1 h and maspin localization was analyzed by immunofluorescence. Nuclei were stained with DAPI. MW markers are indicated on the left side of the images. Images are representative of at least three independent assays. Scale bar: 20 μm