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. 2014 Dec 15;127(24):5303–5316. doi: 10.1242/jcs.157560

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© 2014. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 1. — Eps8 expression is upregulated in SCCs and interacts with FAK. (A) Primary keratinocytes were isolated from mouse tails, and Eps8 expression was compared to a number of SCC lines by western blotting using anti-Eps8 antibody. Anti-β-actin (middle panel) and anti-GAPDH (lower panel) antibodies were used as loading controls. (B) Eps8 expression was compared between SCC cell line 2 (SCC 2) and independent keratinocyte cultures by western blotting using anti-Eps8 antibody (upper panel). Anti-β-actin was used as a loading control (lower panel). (C) Eps8 expression was compared between SCC 2, human SCC cell lines and a human NHK control cell line by western blotting using anti-Eps8 antibody (middle panel). Met, metastatic SCC lines; T, SCC lines from patients who had previously received organ transplants; IC, SCC lines from patients with sporadic primary SCC. FAK expression was analyzed employing anti-FAK antibody (upper panel) and anti-β-actin was used as a loading control (lower panel). (D) Relative Eps8 mRNA expression in mouse and human SCC cell lines was analyzed by qRT-PCR using the ΔΔCt method. GAPDH was used as a control for differences in cDNA input. Results are mean±s.d. *P<0.01; ^#P<0.05 (Student's t-test). (E) FAK expression upon knockdown of endogenous Eps8 by shRNA (shEps8 E lanes; C lanes show control shRNA) or two independent siRNAs (siEps8 #1 and #2; siC, control siRNA) was analyzed using anti-FAK antibody (upper panel). Eps8 knockdown was confirmed by using anti-Eps8 antibody (middle panel) and anti-GAPDH was used as a loading control (lower panel). (F) FAK or Eps8 were immunoprecipitated (IP) from FAK WT and FAK^−/− cell lysates using anti-FAK antibody conjugated to agarose (clone 4.47) (left panel) or anti-Eps8 antibody (right panel), followed by western blot analysis with anti-FAK and anti-Eps8 antibodies (lower panels). Eps8 immunoblotting detects both known isoforms with molecular masses of 97 kDa and 68 kDa. Anti-β-actin was used as a loading control. (G) Focal adhesions were isolated from FAK WT and FAK^−/− cells using hydrodynamic force. Left panels: focal adhesions (arrows) were stained with anti-FAK and anti-paxillin antibodies. Right panels: focal adhesions (solid arrows) were stained with anti-FAK and anti-Eps8. Scale bars: 20 µm.