Loss of T-Cell Protein Tyrosine Phosphatase Induces Recycling of the Platelet-derived Growth Factor (PDGF) -Receptor but Not the PDGF -Receptor

Supplemental Material

This article contains the following supporting material:

• Supplemental Figure S1 - (A) Increased PDGF β-receptor phosphorylation in TC-PTP ko clones. Wt MEFs and the two TC-PTP ko clones were left untreated or were stimulated with 10 ng/ml PDGF-BB and lysed. Receptors were precipitated using WGA-Sepharose and the precipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose filters. Phosphorylated receptors were detected by consecutive immunoblotting with a monoclonal phosphotyrosine antibody (PY99) and PDGF βreceptor antibodies. (B) Decreased rate of receptor degradation in TC-PTP ko clone EFM14. Cells were stimulated with 10 ng/ml PDGF-BB as indicated. Total cell lysates were separated by SDS-PAGE and transferred to nitrocellulose filters, and the amount of PDGF β-receptor was detected by immunoblotting with PDGF β-receptor antiserum. (C) Delayed clearance of cell surface receptors in the TC-PTP ko clone EFM14. Cells were stimulation with 10 ng/ml PDGF-BB, as indicated. The cells were transferred to ice to stop membrane trafficking, and cell surface proteins were labeled using sulfo-NHS-SS-biotin. Following lysis, cell surface proteins were precipitated using streptavidin agarose. Precipitated PDGF β-receptors were detected by immunoblotting with PDGF β-receptor antiserum.

• Supplemental Figure S2 - The delayed degradation of PDGF β-receptors is not due to altered protein synthesis in TC-PTP ko MEFs. Cells were left untreated (upper panel) or were treated for one hour with 20 μg/ml cycloheximide (lower panel), followed by stimulation with 10 ng/ml PDGF-BB, as indicated. Total cell lysates were separated by SDS-PAGE and transferred to nitrocellulose filters, and the amount of PDGF β-receptor was detected by immunoblotting with 2 μg/ml CTβ.

• Supplemental Figure S3 - Ubiquitination of the PDGF β-receptor. Wt (left panels) and TC-PTP ko MEFs (right panels) were stimulated with 10 ng/ml PDGF-BB for the indicated time periods. Following lysis, the PDGF β-receptor was immunoprecipitated, and the precipitated proteins were separated by SDS-PAGE and transferred to PVDF filters. Receptor ubiquitination was detected with the P4G7 monoclonal antibody (top panels) followed by immunoblotting with 2 μg/ml CTβƒn (lower panels)

• Supplemental Figure S4 - Detection of recycled PDGF-BB. TC-PTP ko MEFs were incubated with 2 ng/ml ¹²⁵I-PDGF-BB together with 8 ng/ml unlabeled PDGF-BB for 1 h on ice. Following ligand binding, cells were incubated for five minutes on ice in the absence (black bars) or presence (grey bars) of 3 μM monensin, followed by a five minute incubation at 37ºC to allow for ligand internalization. Internalization was stopped by cooling the cells, and ligand present on the cell surface was removed by a mild acidic wash. The cells were incubated at 37ºC for the indicated time periods, to allow for recycling, in the presence of 100 ng/ml unlabeled PDGF-BB, either in the absence (black bars) or presence (grey bars) of 3 μM monensin. The medium was collected and ¹²⁵I-PDGF-BB was precipitated with trichloroacetic acid. The presence of radiolabeled ligand in the precipitates from triplicate experiments was determined by a γ-counter.

• Supplemental Figure S5 - Trafficking of PDGF α-receptors. (A) Phosphorylated PDGF receptors were precipitated from PAE cells stably expressing PDGF α- or PDGF β-receptors (PAEα and PAEβ, respectively) using WGA agarose. The receptors were subsequently incubated with the indicated amount of recombinant TC-PTP (New England Biolabs) for five minutes at 30ºC. The precipitates receptors were separated by SDS-PAGE and transferred to nitrocellulose filters, and the remaining amount of receptor phosphorylation was detected using phosphotyrosine antibodies. (B) Wt and TC-PTP ko MEFs were stimulated with 10 ng/ml PDGF-AA, as indicated. Total cell lysates were separated by SDS-PAGE and transferred to nitrocellulose filters, and the amount of PDGF α-receptor was detected by immunoblotting with 2 μg/ml CTα. The graph displays the relative decrease in receptor content from separate experiments + SEM. (C) TC-PTP ko MEFs stably re-expressing wt TC-PTP were stimulated with 10 ng/ml PDGF-BB, as indicated. The cells were transferred to ice to stop membrane trafficking, and cell surface proteins were labeled using sulfo-NHS-SS-biotin. Following lysis, cell surface proteins were precipitated using streptavidin agarose. Precipitated PDGF receptors were detected by sequential immunoblotting with 2 μg/ml CTαƒnand 2 μg/ml CTβ.

• Supplemental Figure S6 - Confocal analysis of the subcellular localisation of the PDGF β-receptor and Rab4a-EGFP. Wt MEFs were transiently transfected with Rab4a-EGFP. Serum-starved cells were left untreated or stimulated as indicated with 50 ng/ml PDGF-BB. Following stimulation, the cells were fixed and stained with antibodies against the PDGF β-receptor. The subcellular localisations of the PDGF β-receptor and Rab4a-EGFP were determined using a LSM-510 confocal microscope. The insert represents a magnification of the region inside the white square. The bar represents 10 μm.

• Supplemental Figure S7 - Confocal analysis of the subcellular localisation of the PDGFƒnβ-receptor and Rab11-EGFP. Wt (A) or TC-PTP ko (B) MEFs were transiently transfected with Rab11-EGFP. Serum-starved cells were stimulated as indicated with 50 ng/ml PDGF-BB. Following stimulation, the cells were fixed and stained with antibodies against the PDGF β-receptor. The subcellular localisations of the PDGF β-receptor and Rab11-EGFP were determined using a LSM-510 confocal microscope. The insert represents a magnification of the region inside the white square. The bar represents 10 μm.
• CORRECTION - Corrections to supplemental material and figure legends, post-publication.