mouse anti igf 1r  (Santa Cruz Biotechnology)

Journal: Investigative Ophthalmology & Visual Science

Article Title: Rexinoid NEt-3IB Promotes Resident Macrophage Gene Expression and Mitigates Desiccation-Induced Ocular Surface Disease

doi: 10.1167/iovs.67.4.31

Figure Lengend Snippet: Effects of rexinoid treatment on macrophage gene and protein expression in the desiccation stress dry eye model and potential macrophage-derived IGF-1/IGF-1R signaling axis. ( A ) Heatmaps showing z -score–scaled expression of selected latent-time–associated genes in four treatment groups (NS, None [untreated], Veh, and NEt-3IB) in conjunctival monocyte/macrophage lineage cells sorted from CD45⁺ cells after 5 days of desiccating stress (DS5) ( P adj < 0.001). Genes are organized into five functional categories. The color scale represents z -scores of normalized expression ( blue , low; red , high). These data show desiccating stress–associated shifts in macrophage gene programs and their modulation by NEt-3IB, including enrichment of reparative and growth factor–related genes such as Igf1 . Full gene names for the abbreviations are provided in . ( B ) Representative flow cytometry histograms and quantification validating selected macrophage-associated markers in conjunctival immune cells. ( Left ) Representative histograms of CX3CR1 staining with fluorescence-minus-one (FMO) control, with corresponding quantification of the percentage of CD45⁺CD11b⁺CX3CR1⁺ cells. ( Right ) Representative histograms of IGF-1 staining with FMO control, with corresponding quantification of the percentage of CD45⁺CD11b⁺Mrc1⁺IGF-1⁺ cells. Compared with DS5 no treatment and DS5+vehicle controls, DS5+NEt-3IB increased the proportion ofx CX3CR1⁺ and Mrc1⁺IGF-1⁺ myeloid cells. Each dot represents one biological replicate; bars show mean ± SEM. Statistical significance is indicated as shown: ** P < 0.01; **** P < 0.001; **** P < 0.0001; ns, not significant. ( C ) Representative immunofluorescence images showing IGF-1R/WGA/DAPI staining of wholemount conjunctiva showing surface view ( top ) and βIII-tubulin/IGF-1R/DAPI staining in the cornea ( bottom ). IGF-1R localization is shown because IGF-1 is produced by ocular surface resident macrophages, suggesting a potential macrophage-derived IGF-1/IGF-1R signaling axis acting on ocular surface epithelial and neural compartments during DS. Scale bar : 100 µm.

Article Snippet: The following primary antibodies were used: βIII-tubulin (ab215037; Abcam, Cambridge, UK) and human/mouse IGF-1R (AF-305-NA; R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Derivative Assay, Functional Assay, Flow Cytometry, Staining, Fluorescence, Control, Immunofluorescence, Produced

Journal: Fluids and barriers of the CNS

Article Title: Characterization of choroid plexus in the preterm rabbit pup following subcutaneous administration of recombinant human IGF-1/IGFBP-3.

doi: 10.1186/s12987-023-00460-1

Figure Lengend Snippet: Fig. 2 Impact of postnatal age on serum and CSF levels of IGF-1, and activation of IGF-1R in the ChP of the immature brain. A-D. Levels of IGF-1 in serum (A) and CSF (B) in preterm rabbit pups, evaluated 5 h after s.c. injections of rhIGF-1/rhIGFBP-3 (8 mg/kg) or the corresponding vehicle solution at 0 (5), 24 (29) or 72 (77) hours postnatal age (corresponding time-point for termination within parentheses). The ratio of CSF IGF-1/serum IGF-1 was calculated (C). Linear model displaying the relation between the IGF-1 levels in serum and CSF in 0 (5) hours postnatal age pups (corresponding time-point for termination within parentheses) (D). Dark grey area represents the 95% confidence level. Data are presented as means ± SD (N = 6–9). Differences in IGF-1 concentrations were analyzed using one-way ANOVA with post hoc Tukey test for multiple comparisons of means. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. E-F. Western blot analysis of pERK1/2 and pPKB in ChP from preterm rabbit pups 5 h after s.c. administration of rhIGF-1/rhIGFBP-3 (8 mg/kg) at 0 (5, upper), 24 (29, middle) or 72 h (77, lower) postnatal age (corresponding time-point for termination within parentheses). Data from 3 different experiments are presented as means ± SD (N = 3–4). A representative blot is shown. Differences between groups were analyzed using Student’s t-test. *P ≤ 0.05; NS, not significant

Article Snippet: Sections were then incubated with a cocktail of primary antibodies against IGF-1R (goat polyclonal IgG, 2 μg/ml, diluted in PBS-TX-BSA, AF-305-NA, R&D System, McKinley Place, MN, USA) and IGF-1 (mouse monoclonal IgG, 2 μg/ml, diluted in PBS-TX-BSA, AM33345PU-S, Origene, Herford, Germany) for 16 h at 4 °C.

Techniques: Activation Assay, Western Blot

Journal: Fluids and barriers of the CNS

Article Title: Characterization of choroid plexus in the preterm rabbit pup following subcutaneous administration of recombinant human IGF-1/IGFBP-3.

doi: 10.1186/s12987-023-00460-1

Figure Lengend Snippet: Fig. 4 Angiogenesis-related gene expression and IGF-1R activation in ChPE cells upon stimulation with rhIGF-1/rhIGFBP-3 or rhIGF-1. A. Outline of experimental in vitro setup. B. Western blot analysis of pERK1/2 and pPKB in primary murine ChPE cells following exposure to rhIGF-1/rhIGFBP-3 (300 ng/ml), rhIGF-1 (60 ng/ml) or cell culture media only (Control) for 15 min. Data from are presented as means ± SD (N = 4, pooled samples). C. Heat map, showing respective group mean absolute fold change and hierarchical clustering (N = 3–4), comparing gene expression of 84 genes related to angio genesis using RT2 Profiler PCR Array in primary murine ChPE cells upon stimulation with rhIGF-1/rhIGBP-3 (300 ng/ml), rhIGF-1 (60 ng/ml) or culture medium only (Control) for 2 h. D - I. Individual analysis of selected target genes from the RT2 Profiler PCR Array, Bai1 (D), nitric oxide synthase 3 (Nos3) (E), plasminogen (Plg) (F), C-C motif chemokine 11 (Ccl11) (G), chemokine (C-X-C motif) ligand 2 (Cxcl2) (H) and epidermal growth factor (Egf) (I). Data are presented as means ± SD (N = 4). Differences in gene expression were analyzed using one-way ANOVA with post hoc Tukey for multiple comparisons of means, *P ≤ 0.05. P3-P8, postnatal day 3–8

Article Snippet: Sections were then incubated with a cocktail of primary antibodies against IGF-1R (goat polyclonal IgG, 2 μg/ml, diluted in PBS-TX-BSA, AF-305-NA, R&D System, McKinley Place, MN, USA) and IGF-1 (mouse monoclonal IgG, 2 μg/ml, diluted in PBS-TX-BSA, AM33345PU-S, Origene, Herford, Germany) for 16 h at 4 °C.

Techniques: Gene Expression, Activation Assay, In Vitro, Western Blot, Cell Culture, Control

Journal: Fluids and barriers of the CNS

Article Title: Characterization of choroid plexus in the preterm rabbit pup following subcutaneous administration of recombinant human IGF-1/IGFBP-3.

doi: 10.1186/s12987-023-00460-1

Figure Lengend Snippet: Fig. 6 Schematic illustration of the main findings in the present study. Subcutaneous administration of rhIGF-1/rhIGBP-3 to preterm rabbits (E29), results in a higher level of IGF-1 in serum and CSF at postnatal age at administration of 0 h compared to 24 and 72 h. This is in turn correlated with an increased IGF-1R activation in the ChP at a postnatal age of 5 h, and expression of genes involved in the ChP development. Stimulating ChPE cells with rhIGF-1/ rhIGFBP-3 or rhIGF-1 results in an activation the IGF-1R and an increase in protein synthesis involved in ATP production and mRNA processing

Article Snippet: Sections were then incubated with a cocktail of primary antibodies against IGF-1R (goat polyclonal IgG, 2 μg/ml, diluted in PBS-TX-BSA, AF-305-NA, R&D System, McKinley Place, MN, USA) and IGF-1 (mouse monoclonal IgG, 2 μg/ml, diluted in PBS-TX-BSA, AM33345PU-S, Origene, Herford, Germany) for 16 h at 4 °C.

Techniques: Activation Assay, Expressing

Journal: Journal of Bone Oncology

Article Title: Cav3.2 T-Type calcium channels downregulation attenuates bone cancer pain induced by inhibiting IGF-1/HIF-1α signaling pathway in the rat spinal cord

doi: 10.1016/j.jbo.2023.100495

Figure Lengend Snippet: Increased expression of IGF-1 and IGF-1R and inhibition of IGF-1R reverses the upregulation of Cav3.2 and alleviates pain hypersensitivity in BCP rats. (A) IGF-1 expression increased in the dorsal horn of the spinal cord from day 14 to day 21 in BCP rats compared to sham-operation rats. Upper: representative image of Western blot bands in ipsilateral side of the lumbar enlargement lysates; lower: statistical analysis of protein expression. n = 7. * p < 0.05, two-way ANOVA with Bonferroni’s post hoc test. (B) IGF-1R expression increased in the dorsal horn of the spinal cord from day 3 to day 21 in BCP rats compared to sham-operation rats. Upper: representative image of Western blot bands in ipsilateral side of the lumbar enlargement lysates; lower: statistical analysis of protein expression. n = 7. * p < 0.05, two-way ANOVA with Bonferroni’s post hoc test. (C) Representative immunofluorescent images 14 days after BCP and sham rats. (D) Quantification showed that the percentage of Cav3.2 and IGF-1R colabeled cells increased in the BCP group compared to the sham group. Scale bar = 100 or 50 μm (the partial lumbar enlargements). n = 4. * p < 0.05, *** p < 0.001, unpaired t test. (E) Incubation of IGF-1 for 24 – 48 h in cultured PC12 cells produced an increase in Cav3.2 protein compared to the PBS treatment group. Upper: representative Western blot bands; lower: statistical analysis of protein expression. n = 7. ** p < 0.01, *** p < 0.001, one-way ANOVA with Bonferroni’s post hoc test. (F) JB-1, an antagonist of IGF-1R, prevented the BCP-stimulated accumulation of Cav3.2 compared to NS treatment rats. *** p < 0.001, BCP + NS vs. Sham + NS; # p < 0.05, BCP + JB-1 vs. BCP + NS, unpaired t test. (G and H) Three consecutive days of intrathecal application of JB-1 attenuated mechanical allodynia (G) and thermal hyperalgesia (H) 14 days after BCP. n = 8. *** p < 0.001, BCP + NS vs. Sham + NS; # p < 0.05, ## p < 0.01, ### p < 0.001, BCP + JB-1 vs. BCP + NS, two-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: Based on a standard protocol, free-floating sections were washed in PBS, blocked in 10% normal goat serum with 0.3% Triton X-100 in PBS for 1 h at room temperature, and incubated with one of the following primary antibodies: rabbit anti-Cav3.2 antibody (1: 200, ACC-025, Alomone Labs), mouse anti-IGF-1R β antibody (1:50, sc-390130, Santa Cruz Biotechnology), mouse anti-ionized calcium binding adaptor 1 (IBA1, a microglia marker, 1: 200, ab283319, Abcam), mouse anti-glial fibrillary acidic protein (GFAP, an astrocyte marker, 1: 200, ab4648, Abcam) or mouse anti-neuronal specific nuclear protein (NeuN, a neuronal marker, 1: 200, 66836-1-Ig, Proteintech) in 1% bovine serum albumin (BSA) with 0.3% Triton X-100 at 4 °C for 24 h. Sections were then washed in PBS and incubated with one of the following secondary antibodies at room temperature for 2 h: Alexa Fluor 488 conjugated goat anti-rabbit IgG (1: 100, 111-545-003, Jackson ImmunoResearch Laboratories), Alexa Fluor 568 conjugated goat anti-mouse IgG (1: 200, A-11004, Thermo Fisher Scientific), rinsed in PBS, then dried and covered with anti-fade mounting medium.

Techniques: Expressing, Inhibition, Western Blot, Incubation, Cell Culture, Produced

Journal: Journal of Bone Oncology

Article Title: Cav3.2 T-Type calcium channels downregulation attenuates bone cancer pain induced by inhibiting IGF-1/HIF-1α signaling pathway in the rat spinal cord

doi: 10.1016/j.jbo.2023.100495

Figure Lengend Snippet: Expression of HIF-1α is increased in BCP rats, and knockdown of HIF-1α prevents upregulation of Cav3.2 induced by IGF-1. (A) Nucleoprotein expression of HIF-1α increased from day 7 to day 21 in BCP rats compared to sham operation rats. Upper: representative image of Western blot bands in ipsilateral side of the lumbar enlargement lysates; lower: statistical analysis of protein expression. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001, two-way ANOVA with Bonferroni’s post hoc test. (B) The IGF-1R inhibitor JB-1 reversed the increase in the expression of HIF-1α nucleoprotein expression in the spinal dorsal horn in BCP rats. ** p < 0.01, BCP + NS vs. Sham + NS; ## p < 0.01, BCP + JB-1 vs. BCP + NS, unpaired t test. (C, D) Three consecutive days of intrathecal application of HIF-1α siRNA attenuated mechanical allodynia (C) and thermal hyperalgesia (D) 7 days after BCP. n = 8. *** p < 0.001, BCP + Ctrl siRNA vs. Sham + Ctrl siRNA; ### p < 0.001, BCP + HIF-1α siRNA vs. BCP + Ctrl siRNA, two-way ANOVA with Bonferroni’s post hoc test. (E) Pretreatment with HIF-1α siRNA reversed the increased expression of Cav3.2 induced by IGF-1. n = 6. ** p < 0.01, BCP + Ctrl siRNA vs. Sham + Ctrl siRNA; ### p < 0.001, BCP + HIF-1α siRNA vs. BCP + Ctrl siRNA, unpaired t test. (F) Pretreatment with HIF-1α siRNA reversed the increased expression of Cav3.2 induced by IGF-1. n = 6. ** p < 0.01, IGF-1 + Ctrl siRNA vs. PBS + Ctrl siRNA; ### p < 0.001, IGF-1 + HIF-1α siRNA vs. IGF-1 + Ctrl siRNA, unpaired t test. (G) EMSA showed that nucleus HIF-1α bind to the core promoter region of Cav3.2, and these Cav3.2-labeled probe complexes were abrogated by competition from the unlabeled fragments.

Article Snippet: Based on a standard protocol, free-floating sections were washed in PBS, blocked in 10% normal goat serum with 0.3% Triton X-100 in PBS for 1 h at room temperature, and incubated with one of the following primary antibodies: rabbit anti-Cav3.2 antibody (1: 200, ACC-025, Alomone Labs), mouse anti-IGF-1R β antibody (1:50, sc-390130, Santa Cruz Biotechnology), mouse anti-ionized calcium binding adaptor 1 (IBA1, a microglia marker, 1: 200, ab283319, Abcam), mouse anti-glial fibrillary acidic protein (GFAP, an astrocyte marker, 1: 200, ab4648, Abcam) or mouse anti-neuronal specific nuclear protein (NeuN, a neuronal marker, 1: 200, 66836-1-Ig, Proteintech) in 1% bovine serum albumin (BSA) with 0.3% Triton X-100 at 4 °C for 24 h. Sections were then washed in PBS and incubated with one of the following secondary antibodies at room temperature for 2 h: Alexa Fluor 488 conjugated goat anti-rabbit IgG (1: 100, 111-545-003, Jackson ImmunoResearch Laboratories), Alexa Fluor 568 conjugated goat anti-mouse IgG (1: 200, A-11004, Thermo Fisher Scientific), rinsed in PBS, then dried and covered with anti-fade mounting medium.

Techniques: Expressing, Knockdown, Western Blot, Labeling

Journal: Journal of Bone Oncology

Article Title: Cav3.2 T-Type calcium channels downregulation attenuates bone cancer pain induced by inhibiting IGF-1/HIF-1α signaling pathway in the rat spinal cord

doi: 10.1016/j.jbo.2023.100495

Figure Lengend Snippet: A schematic of IGF-1-mediated upregulation of Cav3.2 T-type channels through HIF-1α in the spinal dorsal horn during the development of BCP. The expression of IGF-1/IGF-1R and HIF-1α were increased in the spinal dorsal horn after BCP. Elevated IGF-1 bound to IGF-1R, subsequently activated HIF-1α and then triggered upregulation of Cav3.2 T-type channels. Potentiated Cav3.2 T-type channels lead to an increase in calcium influx and result in pain hypersensitivity that is BCP–induced.

Article Snippet: Based on a standard protocol, free-floating sections were washed in PBS, blocked in 10% normal goat serum with 0.3% Triton X-100 in PBS for 1 h at room temperature, and incubated with one of the following primary antibodies: rabbit anti-Cav3.2 antibody (1: 200, ACC-025, Alomone Labs), mouse anti-IGF-1R β antibody (1:50, sc-390130, Santa Cruz Biotechnology), mouse anti-ionized calcium binding adaptor 1 (IBA1, a microglia marker, 1: 200, ab283319, Abcam), mouse anti-glial fibrillary acidic protein (GFAP, an astrocyte marker, 1: 200, ab4648, Abcam) or mouse anti-neuronal specific nuclear protein (NeuN, a neuronal marker, 1: 200, 66836-1-Ig, Proteintech) in 1% bovine serum albumin (BSA) with 0.3% Triton X-100 at 4 °C for 24 h. Sections were then washed in PBS and incubated with one of the following secondary antibodies at room temperature for 2 h: Alexa Fluor 488 conjugated goat anti-rabbit IgG (1: 100, 111-545-003, Jackson ImmunoResearch Laboratories), Alexa Fluor 568 conjugated goat anti-mouse IgG (1: 200, A-11004, Thermo Fisher Scientific), rinsed in PBS, then dried and covered with anti-fade mounting medium.

Techniques: Expressing