Journal: Cancers

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

doi: 10.3390/cancers12102895

Figure Lengend Snippet: Figure 3. 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 BS3 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: For gene knockdown experiments, the small interfering RNAs (siRNAs) against FAK, Shc1, c-Src, Arp2, Ezrin, PKA-α, SHP2, HRAS, PTPN12, and shRNA targeting PTPN12 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Activation Assay, In Situ, Western Blot, Phospho-proteomics, Expressing, Control, Transfection

Journal: Cancers

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

doi: 10.3390/cancers12102895

Figure Lengend Snippet: Figure 6. CD99CRIII3 activates PTPN12 to facilitate inhibition of EGFR signaling. (A) In situ PLA performed to assess the interactions between the pairs of molecules indicated in MCF-7 cells. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (B) MDA-MB-231 cells were transfected with PTPN12 siRNA, followed by treatment with EGF (25 ng/mL) with or without CD99CRIII3 (40 µM) for 15 min. (C) MDA-MB-231 cells were treated with EGF and/or CD99CRIII3 in a time-dependent manner. (D) MDA-MB-231 cells were transiently transfected with PTPN12 siRNA or expression plasmids encoding CA-GTPases or WAVE2, ARP2, ROCK2, and Ezrin. Cell lysates were immunoprecipitated with the antibodies indicated. The immunoprecipitates were analyzed by Western blot with the antibodies indicated. (E) The cells stimulated by binding of ligand to its receptor were assayed for activation of small GTPases. β-actin was used as a loading control. (F) For dimerization assay, MDA-MB-231 cells were subjected to BS3 chemical-mediated crosslinking as described above. Cell extracts were assessed by Western blot analysis to determine the dimerization and phosphorylation levels of EGFR. β-actin was used as a loading control. Actin cytoskeleton organization in MDA-MB-231 cells (G) and EGFR endocytosis in MCF-7 cells (H) were determined by IFA as described above. Original magnification of representative images, 600×. Scale bars = 10 µm.

Article Snippet: For gene knockdown experiments, the small interfering RNAs (siRNAs) against FAK, Shc1, c-Src, Arp2, Ezrin, PKA-α, SHP2, HRAS, PTPN12, and shRNA targeting PTPN12 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Inhibition, In Situ, Transfection, Expressing, Immunoprecipitation, Western Blot, Binding Assay, Activation Assay, Control, Phospho-proteomics