Journal: Journal of Nanobiotechnology

Article Title: RVG-targeted extracellular vesicles loaded with echinatin attenuate dopaminergic neurodegeneration via the IGF-2/PI3K/Akt pathway in Parkinson’s disease mice

doi: 10.1186/s12951-025-03997-5

Figure Lengend Snippet: RVG-EVs@Echi attenuate oxidative stress in the in vitro PD models. ( a ) Representative blots and ( b ) quantification of the expression levels of IGF2, P-PI3K, PI3K, P-Akt, Akt, and Nrf2 in MN9D cells. ( c ) Representative blots and ( d ) quantification of Bcl-2, Bax, and the resulting Bcl-2/Bax ratio in MN9D cells. ( e , g ) Representative blots and ( f , h ) quantification showing the effects of the IGF-1R/IR inhibitor NVP-AEW541 in MN9D cells. ( i ) Representative graphs of ROS generation in control or MPP+-treated cells upon RVG-EVs@Echi treatment. Scale bars, 50 μm and 10 μm for the original and magnified images, respectively. ( k ) MitoSOX was used to stain live control or MPP+-treated cells upon treatment with RVG-EVs@Echi. Scale bars, 50 μm and 10 μm for the original and magnified images, respectively. ( j , l ) Quantification of the relative ROS and MitoSOX fluorescence intensity. n = 6 per group. ( m ) Representative flow cytometry histograms and ( n ) quantification of mean fluorescence intensity (MFI) showing intracellular ROS levels in MN9D cells. n = 3 per group. Statistical Analysis: For (a-d): Groups are Control, MPP+, and MPP + + RVG-EVs@Echi. n = 3 per group. * p < 0.05, ** p < 0.01 vs. Control group; # p < 0.05, ## p < 0.01 vs. MPP + group. For (e-h): Groups are Control, MPP + + RVG-EVs@Echi, and MPP + + RVG-EVs@Echi + NVP-AEW541. n = 3 per group. * p < 0.05, ** p < 0.01 vs. Control group; # p < 0.05, ## p < 0.01 vs. MPP + + RVG-EVs@Echi group. For (i-n): Groups are Control, RVG-EVs@Echi, MPP+, and MPP + + RVG-EVs@Echi. * p < 0.05, ** p < 0.01 vs. Control group; # p < 0.05, ## p < 0.01 vs. RVG-EVs@Echi group; & p < 0.05, && p < 0.01 vs. MPP + group

Article Snippet: To assess the effects of RVG-EVs@Echi, MN9D cells were treated with MPP+ (600 μM) or RVG-EVs@Echi (1 μM) for 24 h. For the IGF-1R inhibition experiment, cells were pre-treated with the IGF-1R/IR inhibitor NVP-AEW541 (0.2 μM; MCE, HY-50866) [ ] for 2 h, followed by co-incubation with MPP + and RVG-EVs@Echi for 24 h.

Techniques: In Vitro, Expressing, Control, Staining, Fluorescence, Flow Cytometry

Journal: PLoS ONE

Article Title: Comparative mRNA and miRNA transcriptome analysis of a mouse model of IGFIR-driven lung cancer

doi: 10.1371/journal.pone.0206948

Figure Lengend Snippet: (A) Schematic of doxycycline inducible expression of IGFIR in the mouse lung via the SPC promoter. (B) Unsupervised hierarchical clustering dendrogram of RNA-Seq data from tumor (T, red) and non-transgenic normal lung (N, grey) samples. (C) Volcano plot of log2 fold changes and differential expression p values between tumor and normal lung tissue. (D) Pie chart illustrating percentage of genes up and down-regulated in IGFIR-driven tumors. (E) Dot plots of endogenous murine Igf1r (padj = 5.71E-11) and human IGFIR transgene (padj = 5.65E-249) mRNA expression following mapping to a hybrid genome. (F) Heatmap showing differential expression of markers of AT2 and Club cells as well as subtypes of non-small cell lung cancer. ADC, adenocarcinoma, SCC, squamous cell carcinoma, mADC, mucinous adenocarcinoma. Adjusted p-values for (C) and (E) were obtained from DESeq2.

Article Snippet: To further enrich for tumor cells, a biotinylated anti-human IGF1R antibody (1:10, clone REA271, Miltenyi Biotech) was added to the epithelial enriched fraction for 30 min on ice followed by positive selection and separation as above.

Techniques: Expressing, RNA Sequencing, Transgenic Assay, Quantitative Proteomics

Journal: International Journal of Molecular Medicine

Article Title: miR-589-3p sponged by the lncRNA TINCR inhibits the proliferation, migration and invasion and promotes the apoptosis of breast cancer cells by suppressing the Akt pathway via IGF1R

doi: 10.3892/ijmm.2020.4666

Figure Lengend Snippet: miR-589-3p mimics could partially reverse the effect of IGF1R overexpression on breast cancer cell proliferation and apoptosis. (A) TargetScan predicted the target gene of miR-589-3p. (B and C) Luciferase activity of the miR-589-3p target gene was detected by luciferase assay. (D and E) Cell proliferation of MCF-7 and MDA-MB-231 cells detected by MTT assay. (F and G) Flow cytometry was used to detect MCF-7 and MDA-MB-231 cell apoptosis. All experiments were repeated three times. ** P<0.01 vs. blank or NC; ## P<0.01 vs. IGF1R; ΘΘ P<0.01 vs. IGF1R + miR-589-3p mimic; ΔΔ P<0.01 vs. si-NC; ρ P<0.05 and ρρ P<0.001 vs. siIGF1R; ΓΓ P<0.01 vs. siIGF1R + miR-589-3p inhibitor. IGF1R, insulin-like growth factor 1 receptor; mut, mutant; miR, microRNA; NC, negative control; si, small interfering; wt, wild type.

Article Snippet: Briefly, 1 μ g of pc-TINCR, pcDNA3.2 vector, TINCR small-interfering RNA (siTINCR; cat. no. SR316884; Origene), negative control siRNA (cat. no. SR30004; Origene), miR-589-3p mimic (cat. no. 4464066; Thermo Fisher Scientific, Inc.), miR-589-3p inhibitor (cat. no. 4464084, Thermo Fisher Scientific, Inc.), mimic control (cat. no. 4464058; Thermo Fisher Scientific, Inc.), inhibitor control (cat. no. 4464076; Thermo Fisher Scientific, Inc.), IGF1R (cat. no. RC214928; Origene) or IGF1R small interfering RNA (siIGF1R; cat. no. siB05109161217-1-5; RiboBio) were diluted in 50 μ l of Opti-MEM ® medium (cat. no. 31985062; Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Over Expression, Luciferase, Activity Assay, MTT Assay, Flow Cytometry, Mutagenesis, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: miR-589-3p sponged by the lncRNA TINCR inhibits the proliferation, migration and invasion and promotes the apoptosis of breast cancer cells by suppressing the Akt pathway via IGF1R

doi: 10.3892/ijmm.2020.4666

Figure Lengend Snippet: miR-589-3p mimic inhibited the migratory and invasive abilities of breast cancer cells by down-regulating IGF1R expression. (A-D) Wound healing assay was used to detect the migratory ability of MCF-7 and MDA-MB-231 cells. (E-H) Transwell assay was used to detect the invasive ability of MCF-7 and MDA-MB-231 cells. All experiments were repeated three times. ** P<0.01 vs. NC; ## P<0.01 vs. IGF1R; ΘΘ P<0.01 vs. IGF1R + miR-589-3p mimic; ΔΔ P<0.01 vs. siNC; ρρ P<0.01 vs. si-IGF1R; ΓΓ P<0.01 vs. si-IGF1R + miR-589-3p inhibitor. IGF1R, insulin-like growth factor 1 receptor; miR, microRNA; NC, negative control; si, small interfering.

Article Snippet: Briefly, 1 μ g of pc-TINCR, pcDNA3.2 vector, TINCR small-interfering RNA (siTINCR; cat. no. SR316884; Origene), negative control siRNA (cat. no. SR30004; Origene), miR-589-3p mimic (cat. no. 4464066; Thermo Fisher Scientific, Inc.), miR-589-3p inhibitor (cat. no. 4464084, Thermo Fisher Scientific, Inc.), mimic control (cat. no. 4464058; Thermo Fisher Scientific, Inc.), inhibitor control (cat. no. 4464076; Thermo Fisher Scientific, Inc.), IGF1R (cat. no. RC214928; Origene) or IGF1R small interfering RNA (siIGF1R; cat. no. siB05109161217-1-5; RiboBio) were diluted in 50 μ l of Opti-MEM ® medium (cat. no. 31985062; Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Wound Healing Assay, Transwell Assay, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: miR-589-3p sponged by the lncRNA TINCR inhibits the proliferation, migration and invasion and promotes the apoptosis of breast cancer cells by suppressing the Akt pathway via IGF1R

doi: 10.3892/ijmm.2020.4666

Figure Lengend Snippet: miR-589-3p regulated the activation of Akt pathway by targeting IGF1R in breast cancer cells. (A and B) Reverse transcription-quantitative PCR was used to detect IGF1R and Akt expression levels. (C-E) Western blotting used to detect the protein levels of IGF1R, p-AKT and Akt in MCF-7 cells. (F-H) Western blotting used to detect the protein levels of IGF1R, p-AKT and Akt in MDA-MB-231 cells. All experiments were repeated three times. GAPDH was used as an internal reference. ** P<0.01 vs. NC; ## P<0.01 vs. IGF1R; ΘΘ P<0.01 vs. IGF1R + miR-589-3p mimic; ΔΔ P<0.01 vs. siNC; ρρ P<0.01 vs. siIGF1R; ΓΓ P<0.01 vs. siIGF1R + miR-589-3p inhibitor. IGF1R, insulin-like growth factor 1 receptor; miR, microRNA; NC, negative control; p, phospho; si, small interfering.

Article Snippet: Briefly, 1 μ g of pc-TINCR, pcDNA3.2 vector, TINCR small-interfering RNA (siTINCR; cat. no. SR316884; Origene), negative control siRNA (cat. no. SR30004; Origene), miR-589-3p mimic (cat. no. 4464066; Thermo Fisher Scientific, Inc.), miR-589-3p inhibitor (cat. no. 4464084, Thermo Fisher Scientific, Inc.), mimic control (cat. no. 4464058; Thermo Fisher Scientific, Inc.), inhibitor control (cat. no. 4464076; Thermo Fisher Scientific, Inc.), IGF1R (cat. no. RC214928; Origene) or IGF1R small interfering RNA (siIGF1R; cat. no. siB05109161217-1-5; RiboBio) were diluted in 50 μ l of Opti-MEM ® medium (cat. no. 31985062; Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Activation Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Polyubiquitination of Insulin-like Growth Factor I Receptor (IGF-IR) Activation Loop Promotes Antibody-induced Receptor Internalization and Down-regulation *

doi: 10.1074/jbc.M111.288514

Figure Lengend Snippet: h10H5 induces IGF-IR ubiquitination within the activation loop. A, SK-N-AS cells were serum-starved overnight followed by stimulation with either 100 ng/ml IGF-I or 4 μg/ml h10H5 for the indicated times (in minutes). Cell lysates were immunoprecipitated with 10F5 anti-IGF-IR and Western blotted with anti-ubiquitin (Ub) P4D1 (upper panel) and anti-IGF-IRβ subunit (lower panel; ∼100 kDa) antibodies. B, tandem mass spectrum of the [M + 2H]2+ ion (m/z 626.8779) of the doubly ubiquitinated (K*) peptide K*GGK*GLLPVR from IGF-IR. The spectrum was found during data-dependent analysis of the total immunoprecipitate of IGF-IR on the LTQ-FT hybrid mass spectrometer. The m/z matches within −0.06 Da, and the b ion series (in blue; right to left above the sequence) and y ion series (in green; left to right below the sequence) show complete sequence coverage of this peptide.

Article Snippet: Reagents and Cell Lines Mouse anti-human IGF-IR antibodies h10H5 and 10F5 (Genentech) were generated as described previously ( 30 ).

Techniques: Ubiquitin Proteomics, Activation Assay, Immunoprecipitation, Western Blot, Mass Spectrometry, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Polyubiquitination of Insulin-like Growth Factor I Receptor (IGF-IR) Activation Loop Promotes Antibody-induced Receptor Internalization and Down-regulation *

doi: 10.1074/jbc.M111.288514

Figure Lengend Snippet: siRNA-mediated MDM2, NEDD4, or CBL and CBL-B depletion does not affect h10H5-induced IGF-IR degradation. Non-targeting control (NTC) siRNA (A) or siRNAs against NEDD4 (B), MDM2 (C), or a mixture of CBL and CBL-B (D) were transfected into A549 cells. After 72 h, the cells were treated with 2 μg/ml h10H5 and harvested at the indicated time points. Lysates were separated by SDS-PAGE and analyzed by Western blotting (without prior immunoprecipitation) with anti-IGF-IRβ antibodies to examine down-regulation of endogenous IGF-IR (A–D) and anti-Nedd4 (∼100 kDa) (B), anti-Mdm2 (∼90 kDa) (C), and anti-Cbl and anti-Cbl-b (∼120 kDa) (D) antibodies to verify gene knockdown. β-Actin was used as a loading control.

Article Snippet: Reagents and Cell Lines Mouse anti-human IGF-IR antibodies h10H5 and 10F5 (Genentech) were generated as described previously ( 30 ).

Techniques: Control, Transfection, SDS Page, Western Blot, Immunoprecipitation, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: Polyubiquitination of Insulin-like Growth Factor I Receptor (IGF-IR) Activation Loop Promotes Antibody-induced Receptor Internalization and Down-regulation *

doi: 10.1074/jbc.M111.288514

Figure Lengend Snippet: 2KR and 5KR IGF-IRs exhibit decreased h10H5-induced ubiquitination and receptor down-regulation. A, schematic of the IGF-IR kinase domain (amino acids 944–1264 of human IGF-IR in gray with the activation loop (AL; amino acids 1122–1143) in white) drawn to scale, showing the positions of the lysine to arginine substitutions in mutants 2KR (above) and 5KR (below). B, 2KR and 5KR IGF-IRs are competent for signaling. R− MEFs stably expressing WT, 2KR, or 5KR IGF-IR or empty vector were serum-starved for 5 h and stimulated with 100 ng/ml IGF-I for the indicated time points. Lysates were immunoprecipitated with anti-IGF-IR antibody 10F5 and Western blotted with anti-phosphotyrosine antibody 4G10 (upper panel) or IGF-IRβ subunit (lower panel; loading control). C, 2KR and 5KR IGF-IRs exhibit decreased h10H5-mediated IGF-IR ubiquitination. R− MEFs stably expressing WT or mutant IGF-IR were treated with 4 μg/ml h10H5 (in 10% FBS medium) for the indicated time points. IGF-IR was immunoprecipitated with 10F5 and analyzed by Western blotting with anti-ubiquitin (Ub) antibody P4D1 and IGF-IRβ subunit (loading control). D, 2KR and 5KR IGF-IRs are more resistant to h10H5-induced IGF-IR down-regulation. Cells were treated as in C except for longer time points, and the kinetics of IGF-IR down-regulation were analyzed by direct Western blotting with anti-IGF-IRβ versus β-actin loading control.

Article Snippet: Reagents and Cell Lines Mouse anti-human IGF-IR antibodies h10H5 and 10F5 (Genentech) were generated as described previously ( 30 ).

Techniques: Ubiquitin Proteomics, Activation Assay, Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Control, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Polyubiquitination of Insulin-like Growth Factor I Receptor (IGF-IR) Activation Loop Promotes Antibody-induced Receptor Internalization and Down-regulation *

doi: 10.1074/jbc.M111.288514

Figure Lengend Snippet: 2KR and 5KR mutations affect IGF-I-induced IGF-IR down-regulation and ubiquitination. A, HA-tagged WT, 2KR, and 5KR IGF-IRs are comparable in their signaling capacity. A549 cells stably expressing WT or 2KR or 5KR mutant IGF-IRβ-HA were serum-starved for 5 h and stimulated with 100 ng/ml IGF-I for 10 or 30 min. Transfected IGF-IR was immunoprecipitated with anti-HA antibodies and analyzed by Western blotting with anti-phosphotyrosine antibody 4G10. B, 2KR and 5KR IGF-IRs are more resistant to IGF-I-mediated IGF-IR down-regulation. A549 cells stably expressing HA-tagged WT or mutant IGF-IR were treated with 4 μg/ml h10H5 for 3, 24, or 42 h. The kinetics of receptor down-regulation were analyzed by direct Western blotting with an anti-HA antibody. C, 2KR and 5KR IGF-IRs exhibit decreased IGF-I-mediated IGF-IR ubiquitination. Cells were treated as in A except immunoprecipitates were Western blotted with the anti-ubiquitin (Ub) antibody P4D1. Total IGF-IR (A and C) and β-actin (B) were used as loading controls. Note that the ubiquitination induced by IGF-I is much less extensive than that induced by h10H5 in Fig. 3. The asterisk denotes a nonspecific ubiquitin band immunoprecipitated by the anti-HA antibody (present also in the empty vector control cells).

Article Snippet: Reagents and Cell Lines Mouse anti-human IGF-IR antibodies h10H5 and 10F5 (Genentech) were generated as described previously ( 30 ).

Techniques: Ubiquitin Proteomics, Stable Transfection, Expressing, Mutagenesis, Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Control

Journal: The Journal of Biological Chemistry

Article Title: Polyubiquitination of Insulin-like Growth Factor I Receptor (IGF-IR) Activation Loop Promotes Antibody-induced Receptor Internalization and Down-regulation *

doi: 10.1074/jbc.M111.288514

Figure Lengend Snippet: IGF-IR phosphorylation is required for efficient receptor ubiquitination. A, to-scale schematic of the IGF-IR kinase domain (gray with the activation loop (AL) in white) showing the positions of the K1003R, 3YF (three tyrosines to phenylalanines), and 13KR (all 13 lysines in the kinase domain to arginines (except K1003)) mutants. B, K1003R, 3YF, and 13KR IGF-IRs are defective in IGF-I-mediated signaling. R− MEFs with WT or mutant IGF-IR or empty vector were serum-starved for 5 h and stimulated with 100 ng/ml IGF-I for 10 or 30 min. IGF-IR was immunoprecipitated with 10F5 and analyzed by Western blotting with anti-phosphotyrosine antibody 4G10. C, K1003R, 3YF, and 13KR IGF-IRs exhibit decreased h10H5-mediated receptor ubiquitination. R− MEFs stably expressing WT or mutant IGF-IR were treated with 4 μg/ml h10H5 in complete (10% FBS, which contains IGF-I) medium for 5, 10, or 30 min, and then IGF-IR was immunoprecipitated with 10F5 and Western blotted with anti-ubiquitin (Ub) antibody P4D1. D, as in C except h10H5 treatment was for 1, 4, or 8 h, and the lysates were Western blotted with anti-IGF-IRβ subunit antibody without prior immunoprecipitation to analyze the kinetics of receptor down-regulation. Total IGF-IR (B and C) and β-actin (D) were used loading controls.

Article Snippet: Reagents and Cell Lines Mouse anti-human IGF-IR antibodies h10H5 and 10F5 (Genentech) were generated as described previously ( 30 ).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Activation Assay, Mutagenesis, Plasmid Preparation, Immunoprecipitation, Western Blot, Stable Transfection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Polyubiquitination of Insulin-like Growth Factor I Receptor (IGF-IR) Activation Loop Promotes Antibody-induced Receptor Internalization and Down-regulation *

doi: 10.1074/jbc.M111.288514

Figure Lengend Snippet: Immunofluorescence and quantitative flow cytometry analysis of h10H5 uptake. A, R− MEFs transfected with WT or all the mutant IGF-IRs (or vector alone, which gave no specific signal; not shown) were incubated for 60 min with h10H5 in the presence of lysosomal protease inhibitors to inhibit lysosomal degradation, fixed, and permeabilized, and the total antibody was detected with Cy3-anti-human IgG. Scale bar, 30 μm. See supplemental Fig. 4 for overlays with lysosomes. B, R− MEFs transfected with WT or mutant IGF-IR were incubated on ice for 1 h with Alexa Fluor 488-labeled h10H5, washed, and chased for 20 or 60 min in the presence of lysosomal protease inhibitors, and then any remaining surface antibody was quenched with anti-Alexa Fluor 488 antibodies. Following flow cytometry measurements, the percentage of internalized h10H5 at each time point (after correcting for any losses or incomplete quenching) was calculated from three independent duplicate experiments, each normalized to WT as 100%, with mean and Standard Deviation of the Mean shown. *, p = 0.05; **, p = 0.01; ***, p = 0.001 versus WT (by Student's t test).

Article Snippet: Reagents and Cell Lines Mouse anti-human IGF-IR antibodies h10H5 and 10F5 (Genentech) were generated as described previously ( 30 ).

Techniques: Immunofluorescence, Flow Cytometry, Transfection, Mutagenesis, Plasmid Preparation, Incubation, Labeling, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Polyubiquitination of Insulin-like Growth Factor I Receptor (IGF-IR) Activation Loop Promotes Antibody-induced Receptor Internalization and Down-regulation *

doi: 10.1074/jbc.M111.288514

Figure Lengend Snippet: Cells expressing 2KR or 5KR IGF-IR are more resistant to h10H5-mediated growth inhibition. A, time course of the effect of h10H5 treatment on the viability of MHH-ES-1 cells stably expressing HA-tagged WT or 2KR or 5KR mutant IGF-IR. Cells were treated with 10 μg/ml h10H5 or isotype control anti-gp120 antibody (Ctrl Ab), and cell growth was monitored by the CellTiter-Glo luminescence viability assay at 1, 2, and 3 days with means and Standard Deviation of the Mean of triplicates shown. B, 3-day CellTiter-Glo time point from A normalized to 100% for the gp120 control antibody. Statistical analysis using the Student's t test was performed by comparing mutant versus WT IGF-IR groups treated with h10H5. C, MHH-ES-1 cells expressing WT, 2KR, or 5KR IGF-IR-HA were treated with 2 μg/ml h10H5 for the indicated time points. The kinetics of receptor down-regulation were analyzed by direct Western blotting with anti-HA versus β-actin loading control.

Article Snippet: Reagents and Cell Lines Mouse anti-human IGF-IR antibodies h10H5 and 10F5 (Genentech) were generated as described previously ( 30 ).

Techniques: Expressing, Inhibition, Stable Transfection, Mutagenesis, Control, Viability Assay, Standard Deviation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Polyubiquitination of Insulin-like Growth Factor I Receptor (IGF-IR) Activation Loop Promotes Antibody-induced Receptor Internalization and Down-regulation *

doi: 10.1074/jbc.M111.288514

Figure Lengend Snippet: HCC1419 breast cancer cells exhibit defective h10H5-mediated IGF-IR ubiquitination, internalization, and down-regulation. A, HCC1419 cells were serum-starved overnight and stimulated with 100 ng/ml IGF-I for 0, 10, or 30 min. Endogenous IGF-IR was immunoprecipitated with 10F5 and analyzed with anti-phosphotyrosine antibody 4G10. Total IGF-IRβ subunit was used a loading control. B, HCC1419 and SK-N-AS cells were treated with h10H5 for the time points indicated, and IGF-IR was immunoprecipitated with 10F5 and analyzed with anti-ubiquitin (Ub) antibody P4D1. C, HCC1419 cells were incubated on ice for 30 min with Alexa Fluor 555-h10H5 (top) or Alexa Fluor 555-transferrin (bottom); washed; chased at 37 °C for 0.5, 1, or 5 h; then fixed, and imaged. D, HCC1419 cells were treated with h10H5 for the indicated time points, and IGF-IRβ subunit levels were analyzed by direct Western blotting compared with β-actin loading control.

Article Snippet: Reagents and Cell Lines Mouse anti-human IGF-IR antibodies h10H5 and 10F5 (Genentech) were generated as described previously ( 30 ).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Control, Incubation, Western Blot