Journal: Cancers

Article Title: CD99–PTPN12 Axis Suppresses Actin Cytoskeleton-Mediated Dimerization of Epidermal Growth Factor Receptor

doi: 10.3390/cancers12102895

Figure Lengend Snippet: FAK functions as a critical mediator in EGF-induced activation of Rac1 and RhoA GTPases during EGFR signaling. ( A , C ) MCF-7 cells stimulated by binding of ligand to its receptor were analyzed for activation of small GTPases. Activated GTP-bound Rac1 or RhoA in the cell lysates were determined by immunoblotting with anti-Rac1 or anti-RhoA antibodies. β-actin was used as a loading control. ( B , D ) MDA-MB-231 cells were transfected with CA-FAK or FAK Y397F plasmids and incubated in the presence or absence of 25 ng/mL EGF at 37 °C, 5% CO 2 for 15 min. The interactions between the pairs of molecules indicated were assessed by in situ PLA. *** p < 0.001. ( E ) Activation of small GTPases in MCF-7 cells was determined by immunoblotting. ( F ) EGFR dimerization in MDA-MB-231 cells was assessed by in situ PLA and the experiments were duplicated. ( G ) EGFR endocytosis in MCF-7 cells was determined by IFA as described above. Original magnification of representative images, 600×. Scale bars = 10 μm.

Article Snippet: Active GTPase assay was performed using an active Rac1 or RhoA detection kit (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions.

Techniques: Activation Assay, Binding Assay, Western Blot, Transfection, Incubation, In Situ

Journal: Cancers

Article Title: CD99–PTPN12 Axis Suppresses Actin Cytoskeleton-Mediated Dimerization of Epidermal Growth Factor Receptor

doi: 10.3390/cancers12102895

Figure Lengend Snippet: Modulation of actin polymerization by Rac1/RhoA GTPases is essential for EGF-induced dimerization and endocytosis of EGFR. ( A , E ) The changes in the activation of Rac1/RhoA-mediated signaling were observed in MCF-7 cells. The interactions between the pairs of molecules indicated were assessed by in situ PLA. *** p < 0.001. ( B , F ) To determine EGFR dimerization, MDA-MB-231 cells were subjected to BS 3 chemical-mediated crosslinking, as described above and in the Materials and Methods. Cell extracts were assessed via Western blotting to determine the dimerization and phosphorylation levels of EGFR and the expression levels of indicated proteins. β-actin was used as a loading control. EGFR endocytosis ( C , G ) and actin cytoskeleton organization ( D , H ) in MCF-7 cells transfected with siRNAs specific for ARP2 and Ezrin or plasmids encoding CA-GTPases or CA-FAK. Original magnification of representative images, 600×. Scale bars = 10 μm.

Article Snippet: Active GTPase assay was performed using an active Rac1 or RhoA detection kit (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions.

Techniques: Activation Assay, In Situ, Western Blot, Expressing, Transfection