Chromatin immunoprecipitation with anti-GST antibodies to identify the nuclear localization of GST-sHB-EGF.

<p>Down-regulation of EGFR activity by kinase inhibitor AG1478 and endocytosis inhibitor (PAO) suppressed the association of GST—sHB-EGF with the cyclin D1 promoter in A431 cells. <i>Inp</i>—input nuclear DNA.</p

Chromatin immunoprecipitation with anti-EGFR monoclonal antibody.

<p><b>A</b>, <b>B.</b> Nuclear localization of EGFR following sHB-EGF (A) and H₂O₂ (B) treatment. Increased association of nuclear EGFR and the cyclin D1 promoter region following sHB-EGF treatment is shown. <b>C</b>, Down-regulation of EGFR activity by kinase inhibitor AG1478 suppressed the association of EGFR with the cyclin D1 promoter in A431 cells under sHB-EGF treatment.</p

A time-dependent co-localization of mCherry-sHB-EGF with CGN-ER membranes in A431 cells.

<p>Cells were transfected with p23-EYFP and treated with sHB-EGF during different time periods. AG1478 was added during 60 min of sHB-EGF treatment. (mCherry-sHB-EGF <i>(Red)</i>, CGN-ER membranes <i>(green)</i>; co-localization of mCherry-sHB-EGF with CGN-ER membranes <i>(yellow</i> and <i>white)</i>). Co-localization data was calculated with FIJI software. White scale bar corresponding to 5 um.</p

The EGFR localization in nuclear and cytoplasmic fractions after treatment of A431 cells with sHB-EGF.

<p>Non-nuclear and nuclear cellular extracts were subjected to western-blot analysis after sHB-EGF treatment in the absence or presence of EGFR tyrosine kinase inhibitor AG1478. Down-regulation of EGFR activity by kinase inhibitor AG1478 suppressed the EGFR nuclear localization in A431 cells under sHB-EGF treatment.</p