Sundvall et al. 10.1073/pnas.0708333105. |
Fig. 5. Colocalization of ErbB4 CYT-1 with Rab5 and Rab7. (A) COS-7 cells were transfected with constructs encoding HA-tagged ErbB4 JM-b CYT-1 or ErbB4 JM-b CYT-2 and GFP-Rab5 (A) or GFP-Rab7 (B), and stimulated for 0, 5 or 30 min with 100 ng/ml NRG-1. Localization of ErbB4 (anti-HA antibody; red), Rab5 or Rab7 (GFP; green), and nuclei (DAPI; blue) was visualized by confocal microscopy.
Fig. 6. Down-regulation and degradation of ErbB4 isoforms. (A) COS-7 cells were tranfected with constructs encoding ErbB4 JM-a CYT-1 or ErbB4 JM-a CYT-2. After 6 h, starvation without FCS, cells were stimulated with 50 ng/ml NRG-1 for 0, 5, 15, or 30 min, and incubated on ice in the presence of biotin for 45 min. Biotinylated cell surface-associated ErbB4 was detected by immunoprecipitation with anti-ErbB4 antibody (HFR-1) and Vectastain ABC kit. (B) COS-7 cells were tranfected with constructs encoding ErbB4 JM-b CYT-1, ErbB4 JM-b CYT-2, or EGFR. Cells were labeled with [35S]methionine, washed and lysed 0, 2, 4, and 6 h after labeling. Lysates were immunoprecipitated with anti-EGFR (Santa Cruz Biotechnology) or anti-ErbB4 antibodies (HFR-1) and analyzed by autoradiography of SDS/PAGE gels.
Fig. 7. Coprecipitation of endogenously expressed ErbB4 and Itch. OVCAR-3 cells were starved overnight without FCS, stimulated with 50 ng/ml NRG-1 for 10 min and lysed. Aliquots of cell lysates corresponding to 2 mg of total protein were immunoprecipitated with anti-ErbB4 (HFR-1) (lanes 1 and 2) or anti-PCNA (sc-56; Santa Cruz Biotechnology) (lanes 3 and 4) antibody followed by Western blotting with anti-Itch antibody (BD Biosciences). Membranes were reblotted with anti- ErbB4 (Abcam) (lanes 1 and 2) or anti-PCNA (lanes 3 and 4) antibody.
Fig. 8. Itch monoubiquitinates ErbB4 CYT-1. COS-7 cells expressing Myctagged ErbB4 JM-a CYT-1 and HA-tagged ubiquitin-K48R or ubiquitin-K63R in the presence or absence of Flag-tagged Itch were lysed and the lysates immunoprecipitated with anti-Myc antibody followed by Western blotting with anti-HA antibody. Membranes were reblotted with anti-ErbB4 antibody (Abcam). Itch expression was controlled by Western blotting with anti-Flag antibody.
Fig. 9. Itch regulates endocytosis and degradation of ErbB4 CYT-1. (A) COS-7 cells expressing HA-tagged ErbB4 JM-a CYT-1 or JM-a CYT-2 in the presence or absence of Myc-tagged Itch or Itch C830A were stained with anti-HA antibody (red) and photographed under a fluorescence microscope. (B) COS-7 cells were transfected with constructs encoding HA-tagged ErbB4 JM-b CYT-1 (Left) or HA-tagged ErbB4 JM-b CYT-2 (Right) and Myc-tagged Itch and GFP-Rab7. ErbB4 (anti-HA antibody; red), Rab7 (GFP; green) and nuclei (DAPI; blue) were visualized with confocal microscopy. (C) ErbB4 JM-a CYT-1 and ErbB4 JM-a CYT-2 degradation was analyzed in the presence or absence of Itch or Itch C830A after blocking protein synthesis by cycloheximide. The graphs represent densitometric quantitation of Western data presented in Fig. 4C.
SI Materials and Methods
Analysis of Cell-Surface ErbB4 Down-Regulation by Biotinylation.
COS-7 cells expressing ErbB4 JM-a CYT-1 or JM-a CYT-2 were starved without serum for 6 h, stimulated with NRG-1 (50 ng/ml; R&D Systems) at 37°C for 0, 5, 15, or 30 min in DMEM containing 0.01% BSA. Cell monolayers were washed with ice cold DMEM and incubated on ice with EZ-Linked Sulfo-NHS-LCBiotin (0.5 mg/ml in DMEM; Pierce) for 45 min. Cells washed thrice with ice cold PBS were lysed and 350 μg of total lysates immunoprecipitated with anti-ErbB4 antibody (HFR-1; Neomarkers) as previously described (1). Samples separated in 8% SDS/PAGE gels were analyzed with Vectastain ABC kit to recognize biotinylated ErbB4 (Vector Laboratories). Quantitation of signals exposed to x-ray films was carried out using MCID M5+ software (Imaging Research).Analysis of ErbB4 Half-Life by Metabolic Labeling.
COS-7 cells transiently expressing ErbB4 JM-b CYT-1, JM-b CYT-2, or EGFR were starved for 3 h without serum and additional 2 h in methionine-free medium without serum. The medium was changed to methionine-free medium containing [35S]methionine (20 μCi/ml; MP Biomedicals) for 1 h. Cell monolayers were washed with PBS and incubated in methionine-free medium without serum for 0, 2, 4, or 6 h. Total protein extracts (350 μg) were immunoprecipitated with anti-ErbB4 antibody (HFR-1) or anti-EGFR antibody (sc-03; Santa Cruz Biotechnology), separated in 8% SDS/PAGE gels and analyzed by autoradiography. Quantitation of autoradiographic signals was carried out by using MCID M5+ software.Analysis of ErbB4 Polyubiquitination.
To study the role of lysine-48-linked and lysine-63-linked polyubiquitin chains in Itch-induced ubiquitination of ErbB4, COS-7 cells were transfected to express Myc-tagged ErbB4 JM-a CYT-1 and HA-tagged ubiquitin-K48R or ubiquitin-K63R with or without Itch-Flag. Cells were starved for 12 h and lysed. Five hundred micrograms of total protein was immunoprecipitated with anti-Myc antibody (clone 9 E10; Zymed), separated in 8% SDS/PAGE gels, and blotted with anti-HA (epitope 12CA5; Zymed). Membrane was reblotted with anti-ErbB4 (Abcam).1. Kainulainen V, Sundvall M, Maatta JA, Santiestevan E, Klagsbrun M, Elenius K (2000) J Biol Chem 275:8641-8649.