Journal: Nature

Article Title: Designed endocytosis-inducing proteins degrade targets and amplify signals.

doi: 10.1038/s41586-024-07948-2

Figure Lengend Snippet: Fig. 1 | Design strategies for endocytosis-triggering EndoTags. a, Schema of designed endocytosis mechanisms. Top, design of binding to constitutively cycling receptors at sites that do not overlap with binding sites for natural ligands to avoid competition. Middle, design of binders that trigger endocytosis by eliciting conformational changes in the receptor. The EndoTag binds at two distinct epitopes on the target and actively triggers the conformational change. Bottom, designed endocytosis via receptor clustering. The multivalent EndoTag clusters multiple copies of the target receptor and induces endocytosis. b, Design strategy for sortilin48 and TfR49 EndoTags. c, Cellular uptake of 100 nM AF647-labelled Sort_EndoTags, TfR-EndoTags or LHDB22 scaffold control for 2 h in U-251MG cells. Data were normalized to the 100 nM AF647-labelled LHDB group (no endocytosis). MFI, mean fluorescence intensity. d, Confocal imaging of Sort_EndoTag (red) and lysosomal marker (green, AF488-labelled LysoTracker) after 24 h incubation in U-251MG cells. e, Design strategy for

Article Snippet: Anti-LAMP2A antibody (Abcam ab18528), goat anti-rabbit- IgG Alexa FluorTM 488 secondary antibody (Thermo Fisher A-11034), anti PD-L1 (sc-518027), anti beta-actin (sc-47778), goat anti-mouse IgG H&L (HRP) (Abcam, ab205719); 800CW goat-anti-mouse or goatanti-rabbit (LI-COR 926-32211), rabbit anti-CTLA4 E1V6T Cell Signaling Technologies (96399) Validation Alexa Fluor® 647 anti-human EGFR Antibody (Biolegend, 352918): Verified Reactivity to Human, FC - Quality tested PE anti-human CD222 (IGF2R) Recombinant Antibody (Biolegend, 364204): Verified Reactivity to Human, ICFC, FC - Quality tested EGFR Monoclonal Antibody (clone 199.12, Invitrogen, AHR5072): Target Species: Human, applications for Immunocytochemistry, Immunofluorescence, Immunoprecipitation, Western Blot IGF2R Antibody Alexa647 (clone 2G11, Novus, NB300-514AF647): Reactivity Hu, Mu, Rt, Bv, Pm; Applications WB, ELISA, Flow, ICC/IF, IHC, IP, CyTOF-ready Human PD-L1 Alexa Fluor® 647-conjugated Antibody (Rndsystems, FAB1562R): Species Reactivity Human, Detects human PD-L1/B7H1 in direct ELISAs.

Techniques: Binding Assay, Control, Fluorescence, Imaging, Marker, Incubation

Journal: Nature

Article Title: Designed endocytosis-inducing proteins degrade targets and amplify signals.

doi: 10.1038/s41586-024-07948-2

Figure Lengend Snippet: Fig. 3 | Clearance of soluble proteins by IGF2R pLYTACs. a, Schema for the use of soluble pLYTACs with IGF_EndoTags. b, Cellular uptake of LHDB–AF647 via LHDA–IGF_EndoTags in Jurkat cells. Cells were incubated with 33 nM LHDB– AF647 with or without 1 μM LHDA–IGF_EndoTags for 24 h, washed twice with cold PBS and analysed by flow cytometry. c, Remaining supernatant LHDB– AF647 levels in Jurkat cells. Jurkat cells were incubated with 100 nM LHDB– AF647 with or without 500 nM LHDA–IGF_EndoTags. At timepoints 24 h and 48 h, the cells were pelleted down, and IgG in the supernatant was quantified using a Neo2 plate reader. IgG level was normalized to the IgG-alone control group. d, Cellular uptake of IgG–AF647 via protein G–IGF_EndoTags in K562 cells. Cells were incubated with 33 nM IgG–AF647 with or without 1 μM protein G–IGF_EndoTag3 for 24 h, washed twice with cold PBS and analysed by flow cytometry. The fold change in MFI was calculated by normalizing to the

Article Snippet: Anti-LAMP2A antibody (Abcam ab18528), goat anti-rabbit- IgG Alexa FluorTM 488 secondary antibody (Thermo Fisher A-11034), anti PD-L1 (sc-518027), anti beta-actin (sc-47778), goat anti-mouse IgG H&L (HRP) (Abcam, ab205719); 800CW goat-anti-mouse or goatanti-rabbit (LI-COR 926-32211), rabbit anti-CTLA4 E1V6T Cell Signaling Technologies (96399) Validation Alexa Fluor® 647 anti-human EGFR Antibody (Biolegend, 352918): Verified Reactivity to Human, FC - Quality tested PE anti-human CD222 (IGF2R) Recombinant Antibody (Biolegend, 364204): Verified Reactivity to Human, ICFC, FC - Quality tested EGFR Monoclonal Antibody (clone 199.12, Invitrogen, AHR5072): Target Species: Human, applications for Immunocytochemistry, Immunofluorescence, Immunoprecipitation, Western Blot IGF2R Antibody Alexa647 (clone 2G11, Novus, NB300-514AF647): Reactivity Hu, Mu, Rt, Bv, Pm; Applications WB, ELISA, Flow, ICC/IF, IHC, IP, CyTOF-ready Human PD-L1 Alexa Fluor® 647-conjugated Antibody (Rndsystems, FAB1562R): Species Reactivity Human, Detects human PD-L1/B7H1 in direct ELISAs.

Techniques: Incubation, Flow Cytometry, Control

Journal: Journal of Cancer Prevention

Article Title: Kaempferol Downregulates Insulin-like Growth Factor-I Receptor and ErbB3 Signaling in HT-29 Human Colon Cancer Cells

doi: 10.15430/JCP.2014.19.3.161

Figure Lengend Snippet: Effect of kaempferol on the expression of insulin-like growth factor (IGF)-II and heregulin (HRG) and the phosphorylation of Akt and extracellular regulated kinase (ERK)- 1/2. HT-29 cells were serum-starved and treated with various concentrations (0, 20, 40, or 60 μmol/L) of kaempferol. (A) At 24 hours after treatment, 24 hour-conditioned media were collected and subjected to Western blot analysis with anti-IGF-II antibody. Media loaded onto the gel was adjusted for equivalent cell numbers. (B) At the indicated time point after kaempferol treatment, total cell lysates were prepared and subjected to Western blot analysis with their relevant antibodies. (C, D) At 2 hours after kaempferol treatment, total cell ly-sates were prepared. (C) Total cell lysates were subjected to Western blot analysis with the relevant antibodies. (D) Total cell lysates were incubated with immobilized P-ERK- 1/2 antibody overnight (4 ° C). After centrifugation, the immunoprecipitated proteins were incubated with Elk-1 (an ERK-1/2 substrate) and ATP for 30 minutes (30 ° C). The resulting P-Elk-1 was analyzed by Western blot analysis with an anti-P-Elk antibody. IP, immunoprecipitation; WB, western blot analysis.

Article Snippet: Kaempferol, essentially fatty acid-free bovine serum albumin, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), 7-amino-actinomycin D (7-AAD), phosphatidylinositol, transferrin and anti-β-actin antibody were obtained from Sigma (St. Louis, MO, USA); selenium and DMEM/Ham’s F-12 nutrient mixture (DMEM/F-12 were purchased from Gibco BRL (Gaithersburg, MD, USA)); [methyl- 3 H]thymidine, protein A-Sepharose and horse-radish peroxidase-conjugated anti-rabbit and anti-mouse IgG were from Amersham (Arlington Heights, IL, USA); antibodies against HRG, IGF-IRβ, p85 and ErbB3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against Akt, phospho (P)-Akt, ERK-1/2 and P-ERK-1/2 were from Cell Signaling Technology (Beverly, MA, USA); anti-phosphotyrosine-RC20 (PY20) antibody linked to horse-radish peroxidase was purchased from BD Transduction Laboratories (Palo Alto, CA, USA); anti-IGF-II antibody was from Amano International Enzyme (Troy, VA, USA); recombinant human HRG-β and recombinant human IGF-II were purchased from R&D Systems, (Minneapolis, MN, USA); phycoerythrin-conjugated Annexin V was from BD Pharmingen, (Franklin Lake, NJ, USA); and [ γ - 32 P]ATP was obtained from PerkinElmer Life and Analytical Sciences, Inc. (Boston, MA, USA).

Techniques: Expressing, Phospho-proteomics, Western Blot, Incubation, Centrifugation, Immunoprecipitation

Journal:

Article Title: IGF-2 and FLT-1/VEGF-R1 mRNA levels reveal distinctions and similarities between congenital and common infantile hemangioma

doi: 10.1203/PDR.0b013e318163a243

Figure Lengend Snippet: IGF-2 protein localized in relation to blood vessels in common and congenital hemangioma. A–C) CD31 immunostaining of frozen sections highlights blood vessels in proliferating common hemangioma (A), NICH (B) and RICH (C). Nuclei are stained blue with DAPI. D–E) Common hemangioma sections double-labeled with anti-IGF2 (green) and anti-CD31 (red). Nuclei stained with DAPI (blue). Panel D, a proliferating phase hemangioma while Panel E, involuting phase hemangioma. F) RICH double-labeled with anti-IGF2 (green) and anti-CD31 (red). G) NICH stained with anti-IGF2 (green). H) Pyogenic granuloma stained with anti-IGF2 (green) as negative control. Scale bars are 50 µm. Panel A is 200X magnification; Panels B, C, G and H, 100X magnification; Panels D–F, 300X magnification.

Article Snippet: Indirect immunofluorescence Cryosections ( 4 μm ) of the tumors (proliferating and involuting infantile hemangioma), RICH, NICH and pyogenic granulomas were fixed with cold acetone for 15 min. After washing for 5 min in PBS, and incubating with species appropriate blocking serum, the following primary antibodies were used: mouse anti-human IGF2 mAb 1:50 (R&D Systems, USA), mouse anti-human CD31 mAb 1:50 (DAKO, Carpinteria, CA, USA), goat anti-human CD31 antibody 1:50 (Santa Cruz Biotechnology Inc., CA, USA).

Techniques: Immunostaining, Staining, Labeling, Negative Control