phosphorylated igf1r  (Santa Cruz Biotechnology)

Journal: Neoplasia (New York, N.Y.)

Article Title: Secreted insulin-like growth factor binding protein 5 functions as a tumor suppressor and chemosensitizer through inhibiting insulin-like growth factor 1 receptor/protein kinase B pathway in acute myeloid leukemia.

doi: 10.1016/j.neo.2023.100952

Figure Lengend Snippet: Fig. 3. IGFBP5 overexpression in AML cells inhibits IGF1R activity by secreting IGFBP5. (A) ELISA qualification of IGFBP5 concentrations in the culture supernatants of IGFBP5-transduced U937 and THP1 cells. (B) Immunoblotting of p-IGF1R and total IGF1R protein in IGFBP5-transduced U937 and THP1 cells. GAPDH was used as a loading control. Blank: the untreated parental cells; Control: the empty vector -transduced cells; IGFBP5-OE: the IGFBP5 -transduced cells. Sta tistical significance was determined by ANOVA with Tukey’s multiple com parison test. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.

Article Snippet: The primary antibodies used were as follows: IGFBP5 (Cat No. 10941; CST), IGF1R (Cat No. 9750; CST), phosphorylated (p)-IGF1R β/Irβ (Tyr1131/ Tyr1146) (Cat No. 80732; CST), phosphatase and tensin homolog deleted on chromosome ten (PTEN, Cat No. 9188; CST), PI3K (Cat No. 4257; CST), p-PI3K (Tyr458) (Cat No. 17366; CST), AKT (Cat No. 9272; CST), p-AKT (Ser473) (Cat No. 4060; CST), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, AF0006; Beyotime), p21 (Cat No. 2847; CST), cyclindependent kinase 2 (CDK2, Cat No. 18048; CST), cyclin-dependent kinase 4 (CDK4, Cat No. 12790; CST), cyclin-dependent kinase 6 (CDK6, Cat No. 13331; CST), Cyclin D1 (Cat No. 55506; CST), Cyclin E1 (Cat No. 20808; CST), β-actin (AA128; Beyotime).

Techniques: Over Expression, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Plasmid Preparation

Journal: Neoplasia (New York, N.Y.)

Article Title: Secreted insulin-like growth factor binding protein 5 functions as a tumor suppressor and chemosensitizer through inhibiting insulin-like growth factor 1 receptor/protein kinase B pathway in acute myeloid leukemia.

doi: 10.1016/j.neo.2023.100952

Figure Lengend Snippet: Fig. 4. Treatment with rhIGFBP5 suppresses AML cell proliferation. (A) Representative flow plot of cell proliferation analysis by Ki-67 staining after rhIGFBP5 treatment (n=3). U937 or THP1 cells were treated with 500 ng/mL rhIGFBP5 protein for 72 h. The quantification is shown on the right. (B) Treatment with 500 ng/mL rhIGFBP5 protein induces cell apoptosis in U937 and THP1 cells. After treatment for 72 h, the cells were stained with 7-AAD and Annexin V-APC and analyzed by Flow Cytometry. Shown are representative flow cytometry plots. The quantification is shown on the right. (C) Treatment with 500 ng/mL rhIGFBP5 protein blocks G1-S phase transition in U937 and THP1 cells. After treatment for 72 h, the cells were stained with PI and analyzed by Flow Cytometry. Shown are representative Flow Cytometry plots. The quantification is shown on the right. (D) Immunoblotting of p-IGF1R and total IGF1R protein in U937 and THP1 cells after treatment with 500 ng/mL rhIGFBP5 for 72 h. GAPDH was used as a loading control. Control: the untreated cells; rhIGFBP5: the cells treated with 500 ng/mL rhIGFBP5 protein. Statistical significance was determined by unpaired Student’s t test. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.

Article Snippet: The primary antibodies used were as follows: IGFBP5 (Cat No. 10941; CST), IGF1R (Cat No. 9750; CST), phosphorylated (p)-IGF1R β/Irβ (Tyr1131/ Tyr1146) (Cat No. 80732; CST), phosphatase and tensin homolog deleted on chromosome ten (PTEN, Cat No. 9188; CST), PI3K (Cat No. 4257; CST), p-PI3K (Tyr458) (Cat No. 17366; CST), AKT (Cat No. 9272; CST), p-AKT (Ser473) (Cat No. 4060; CST), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, AF0006; Beyotime), p21 (Cat No. 2847; CST), cyclindependent kinase 2 (CDK2, Cat No. 18048; CST), cyclin-dependent kinase 4 (CDK4, Cat No. 12790; CST), cyclin-dependent kinase 6 (CDK6, Cat No. 13331; CST), Cyclin D1 (Cat No. 55506; CST), Cyclin E1 (Cat No. 20808; CST), β-actin (AA128; Beyotime).

Techniques: Staining, Flow Cytometry, Sublimation, Western Blot, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Secreted insulin-like growth factor binding protein 5 functions as a tumor suppressor and chemosensitizer through inhibiting insulin-like growth factor 1 receptor/protein kinase B pathway in acute myeloid leukemia.

doi: 10.1016/j.neo.2023.100952

Figure Lengend Snippet: Fig. 5. Intracellular IGFBP5 has no effect on AML cell proliferation. (A) Prediction of a secretory signal peptide containing 20 amino acid residues in N-terminus of IGFBP5 by SignaIP 4.0. (B) ELISA qualification of IGFBP5 con centrations in the culture supernatants of mtIGFBP5-transduced U937 and THP1 cells. (C) Immunoblotting of IGFBP5, p-IGF1R and total IGF1R protein in mtIGFBP5- transduced U937 and THP1 cells. GAPDH was used as a loading control. (D) The ectopical expression of mtIGFBP5 in U937 and THP1 cells has no effect on AML cell proliferation (n=3). Cell viability was measured by CCK-8 assay at the indicated time points (day 1, day 2, day 3, day 4, day 5). Blank: the untreated parental cells; Control: the empty vector-transduced cells; mtIGFBP5-OE: the mtIGFBP5-transduced cells. Statistical significance was determined by ANOVA with Tukey’s multiple comparison test. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.

Article Snippet: The primary antibodies used were as follows: IGFBP5 (Cat No. 10941; CST), IGF1R (Cat No. 9750; CST), phosphorylated (p)-IGF1R β/Irβ (Tyr1131/ Tyr1146) (Cat No. 80732; CST), phosphatase and tensin homolog deleted on chromosome ten (PTEN, Cat No. 9188; CST), PI3K (Cat No. 4257; CST), p-PI3K (Tyr458) (Cat No. 17366; CST), AKT (Cat No. 9272; CST), p-AKT (Ser473) (Cat No. 4060; CST), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, AF0006; Beyotime), p21 (Cat No. 2847; CST), cyclindependent kinase 2 (CDK2, Cat No. 18048; CST), cyclin-dependent kinase 4 (CDK4, Cat No. 12790; CST), cyclin-dependent kinase 6 (CDK6, Cat No. 13331; CST), Cyclin D1 (Cat No. 55506; CST), Cyclin E1 (Cat No. 20808; CST), β-actin (AA128; Beyotime).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Control, Expressing, CCK-8 Assay, Plasmid Preparation, Comparison

Journal: Neoplasia (New York, N.Y.)

Article Title: Secreted insulin-like growth factor binding protein 5 functions as a tumor suppressor and chemosensitizer through inhibiting insulin-like growth factor 1 receptor/protein kinase B pathway in acute myeloid leukemia.

doi: 10.1016/j.neo.2023.100952

Figure Lengend Snippet: Fig. 7. Secreted IGFBP5 inhibits IGF1R-mediated PI3K/AKT pathway. (A) Immunoblotting of PI3K/AKT signaling pathway molecules in IGFBP5-transduced THP1 and U937 cells. GAPDH was used as a loading control. (B) Immuno blotting of some cell cycle-related proteins in IGFBP5-transduced THP1 and U937 cells. β-actin was used as a loading control. Blank: the untreated parental cells; Control: the empty vector-transduced cells; IGFBP5-OE: the IGFBP5-transduced cells.

Article Snippet: The primary antibodies used were as follows: IGFBP5 (Cat No. 10941; CST), IGF1R (Cat No. 9750; CST), phosphorylated (p)-IGF1R β/Irβ (Tyr1131/ Tyr1146) (Cat No. 80732; CST), phosphatase and tensin homolog deleted on chromosome ten (PTEN, Cat No. 9188; CST), PI3K (Cat No. 4257; CST), p-PI3K (Tyr458) (Cat No. 17366; CST), AKT (Cat No. 9272; CST), p-AKT (Ser473) (Cat No. 4060; CST), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, AF0006; Beyotime), p21 (Cat No. 2847; CST), cyclindependent kinase 2 (CDK2, Cat No. 18048; CST), cyclin-dependent kinase 4 (CDK4, Cat No. 12790; CST), cyclin-dependent kinase 6 (CDK6, Cat No. 13331; CST), Cyclin D1 (Cat No. 55506; CST), Cyclin E1 (Cat No. 20808; CST), β-actin (AA128; Beyotime).

Techniques: Western Blot, Control, Plasmid Preparation

Journal: Cell reports methods

Article Title: Grabody B, an IGF1 receptor-based shuttle, mediates efficient delivery of biologics across the blood-brain barrier.

doi: 10.1016/j.crmeth.2022.100338

Figure Lengend Snippet: Figure 1. IGF1R is enriched in the brain and the brain vasculature (A) Representative western blots illustrating the differences in the band intensities of IGF1R in each organ (n = 3). Note the low expression of IGF1R in the colon, liver, and heart below the detection limit. b-Actin was used as a loading control. (B) Percentage intensity of IGF1R-immunoreactive bands normalized to the band intensity of the b-actin. n = 3 mice. (C) IGF1R protein expression in the brain vascular or parenchymal fractions from mice. The bands are immuno-reactive to CD31, IGF1R, and b-actin in the vascular and parenchymal fractions (n = 3). (D) Quantification of the intensity of CD31-immunoreactive bands in (C). (E) Quantification of the intensity of IGF1R-immunoreactive bands in (C). The intensity of CD31- and IGFR-positive bands was normalized to b-actin (D and E). (F) Representative images of human temporal lobes immuno-stained with anti-IGF1R antibody. Brain sections from three subjects showed clear expression of IGF1R in brain cells (a, e) and capillaries. BECs (b–d, f–h) are shown in brown with arrows.

Article Snippet: Finally, the proteins were blotted with antibodies against phosphorylated-Akt (Cell Signaling, 9271S), total Akt (Cell Signaling, 9272S), phosphorylated-IGF1R (Cell Signaling, 3024S), total IGF1R (Cell Signaling, 3018S), and b-actin (Santa Cruz, Sc-47778).

Techniques: Western Blot, Expressing, Control, Staining

Journal: Cell reports methods

Article Title: Grabody B, an IGF1 receptor-based shuttle, mediates efficient delivery of biologics across the blood-brain barrier.

doi: 10.1016/j.crmeth.2022.100338

Figure Lengend Snippet: Figure 2. Grabody B does not interfere with the biological actions of IGF1R (A) Schematic representation of M30103 and B30104. B30104 was constructed as a monovalent, bispecific antibody with an anti-a-Syn antibody (M30103) and Grabody B in an scFv format. (B) Western blot of key IGF1R-signaling components after co-treatment of MCF7 cells with IGF1 and four different antibodies. Note the clear induction of the phosphorylation of Akt and IGF1R in the presence of IGF1 and the reduction by A12, a neutralizing anti-IGF1R antibody. In contrast, little if any alteration by M30103 or B30104 was observed. (C and D) Relative fold intensity of pIGF1R-, IGF1R-, pAKT- and AKT-immunoreactive bands in (B). The bands were normalized to the band intensity of the b-actin. n = 3 mice. (E) IGF1-induced MCF7 proliferation with varying concentration of four antibodies. M30103 and B30104 had a minimal effect, whereas two neutralizing anti- IGF1R antibodies (A12, red and MKJP2, magenta) inhibited cell growth. n = 3 cases. Data are presented as the mean ± SD.

Article Snippet: Finally, the proteins were blotted with antibodies against phosphorylated-Akt (Cell Signaling, 9271S), total Akt (Cell Signaling, 9272S), phosphorylated-IGF1R (Cell Signaling, 3024S), total IGF1R (Cell Signaling, 3018S), and b-actin (Santa Cruz, Sc-47778).

Techniques: Construct, Western Blot, Phospho-proteomics, Concentration Assay

Journal: Cell reports methods

Article Title: Grabody B, an IGF1 receptor-based shuttle, mediates efficient delivery of biologics across the blood-brain barrier.

doi: 10.1016/j.crmeth.2022.100338

Figure Lengend Snippet: Figure 5. B30104 reduces pathological a-Syn burden more effectively than M30103 (A) Schematic of the PFF-injection efficacy test (modified from Tran et al., 2014). One week following the stereotaxic injection, the animals received 15 mg/kg of hIgG or M30103 or 17.55 mg/kg of B30104 (black triangle). The behavior of the animals was analyzed after 180 days with 24 doses and sacrificed to assess the neuro- pathology. (B) Representative images of brain sections stained with anti-p-a-Syn antibody. Note the increased p- a-Syn burden 180 dpi in both the prefrontal cortex (globular or cellular type signal) and substantia nigra (cellular and fibrotic signal) of the hIgG-treated mice in contrast to the reduced p-a-Syn immunoreac- tivity of the M30103- or B30104-treated brains. B30104 induced further reduction of the p-a-Syn signal in both brain areas compared with M30103. Very little signal was observed in the non-PFF-in- jected cases. Scale bar, 100 mm. (C and D) Quantification of the p-a-Syn-immunore- activity in (B) as the diffused signal filling an entire cell (C, quantified as p-a-Syn-positive cells) and the number of more dense intracellular inclusions similar to Lewy bodies (LB, dense cellular inclusions filled with filamentous a-Syn aggregates found in PD brains) and Lewy neurites (LN, neurites con- taining filamentous similar to LB found in PD brains) (D, quantified as LB/LN-like inclusions). n = 6 mice. One-way ANOVA with Tukey’s post hoc; *p < 0.05, **p < 0.01, ****p < 0.0001. Data are presented as mean ± SD; n = 6.

Article Snippet: Finally, the proteins were blotted with antibodies against phosphorylated-Akt (Cell Signaling, 9271S), total Akt (Cell Signaling, 9272S), phosphorylated-IGF1R (Cell Signaling, 3024S), total IGF1R (Cell Signaling, 3018S), and b-actin (Santa Cruz, Sc-47778).

Techniques: Injection, Staining, Activity Assay

Journal: bioRxiv

Article Title: Human relevant exposure to di-n-butyl phthalate tampers with the ovarian insulin-like growth factor 1 system and disrupts folliculogenesis in young adult mice

doi: 10.1101/2023.03.15.532792

Figure Lengend Snippet: Young adult cycling female CD-1 mice were treated daily with vehicle (oil) or DBP and their ovaries processed for qPCR as described in Materials and Methods. The expression of Igf1 ( A ), Igf2 ( B ), and Igf1r ( C ) was normalized to housekeeping genes Actb and Tbp. Data are presented as mean normalized relative expression ± SEM. Asterisks (*) indicate statistical significance at the p ≤ 0.05 level (n=8-10 mice/treatment).

Article Snippet: Slides were then incubated with primary anti-phosphorylated IGF1R antibody (Cat#3021, Cell Signaling Technology Inc.; Danvers, MA) at 2.11 μg/ml or protein concentration matched Isotype (overnight at 4°C).

Techniques: Expressing

Journal: bioRxiv

Article Title: Human relevant exposure to di-n-butyl phthalate tampers with the ovarian insulin-like growth factor 1 system and disrupts folliculogenesis in young adult mice

doi: 10.1101/2023.03.15.532792

Figure Lengend Snippet: Young adult cycling female CD-1 mice were treated daily with vehicle (oil) or DBP and their ovaries processed for immunohistochemical staining with anti-pIGF1R and its matched IgG (negative control) as described in Materials and Methods. Representative images from ovarian sections show: no staining for the antibody protein-matched isotype negative control ( A ), expression of IGF1R (depicted by brown staining) in vehicle control ( B ) or DBP 10 μg/kg/day ( C ), 100 μg/kg/day ( D ), and 1000 mg/kg/day ( E ) ovaries. Positive staining was observed in atretic follicles ( F ).

Article Snippet: Slides were then incubated with primary anti-phosphorylated IGF1R antibody (Cat#3021, Cell Signaling Technology Inc.; Danvers, MA) at 2.11 μg/ml or protein concentration matched Isotype (overnight at 4°C).

Techniques: Immunohistochemical staining, Staining, Negative Control, Expressing, Control