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. 2011 Jun 6;208(6):1229–1242. doi: 10.1084/jem.20101880

Figure 5.

Figure 5.

tPA interacts with and activates EGFR to mediate its antiapoptotic effects in OLs. (a) 50 µg of total proteins from lysates of untreated (control) or biotinylated tPA (Biot-tPA)–treated mouse OLs (10 µg/ml) or purified Biot-tPA were subjected to immunoprecipitation using anti-EGFR antibody followed by detection with either peroxidase-coupled avidin (Avidin perox) or with anti-EGFR antisera (αEGFR). As a control, the same procedure was performed by omitting the anti-EGFR antiserum (No Ab). Representative images of immunoblots from three individual experiments are presented. Numbers indicate molecular mass of standard proteins in kilodaltons. Single chain or double chain forms of tPA are indicated as sctPA or dctPA, respectively. (b) Cultured mouse OLs were treated with 10 µg/ml tPA for 24 h or not treated (control). Photomicrographs show representative fields after A2B5 (green) and phosphorylated EGFR (red) immunostaining or the merged images. (c) The phosphorylated form of EGFR was immunodetected in 20 µg of total proteins from OLs subjected to TFD in the presence of 10 µg/ml tPA for the indicated length of time. Anti–α-tubulin was used as a control. Representative images from three individual experiments are presented. Bar, 50 µm. (d–g) Mouse OL death (percentage) was estimated by measuring the LDH release in the media after 24 h of TFD (black bar). (d) Cotreatment with 10 µg/ml tPA (light gray bar) in the presence of 0.25–5 µM AG1487 (dark gray bars; mean + SEM; n = 12). * and **, significantly different from TFD, P < 0.05 and P < 0.01, respectively; #, significantly different from 1 µM AG1487 cotreatment, P < 0.05. (e) Cotreatment with 10 ng/ml EGF with or without an inhibitor of the kinase activity of 5 µM EGFR (AG1487; mean + SEM; n = 8, three independent plates). #, significantly different from TFD + EGF, P < 0.05. (f) Cotreatment with 10 µg/ml of rat recombinant WT or EGF domain–deleted (ΔEGF) tPA (f) or cotreatment with 1–50 µg/ml of a recombinant peptide corresponding to the EGF domain of tPA (rEGF domain) or protein from uninduced bacterial cultures (uninduced) as a control (g; mean + SEM; n = 8, three independent plates). *, significantly (P < 0.05) different from TFD; #, significantly (P < 0.05) different from TFD + wttPA. (h) Diagram of the different mutant forms of tPA used in this study. (i) Mouse neuronal death (percentage) was estimated by measuring the LDH release in the media after 24 h of serum deprivation (black bar) and cotreatment with 20 µg/ml tPA (light gray bar) with or without 5 µM AG1487 (dark gray bars; mean + SEM; n = 12). **, significantly different from TFD, P < 0.01.