anti igf1r  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Arp2/3 complex-dependent actin remodeling is required for efficient RSV uncoating in A549 cells

doi: 10.64898/2026.03.11.711013

Figure Lengend Snippet: (A) Representative confocal microscopy images of RSV-GFP (MOI = 1) bound to A549 WT and Arp2 KO17 cells. Cells were incubated with virus or mock-infected for 1 h on ice, washed to remove unbound virus, fixed, and stained with Palivizumab (anti-RSV-fusion protein, in pseudocolor cyan), Hoechst 33342 (cell nuclei, in pseudocolor green), and WGA (wheat germ agglutinin-Alexa Flour 647, magenta). Images were acquired on a spinning-disk microscope and are shown as maximum-intensity projections of respective channels. Scale bar, 50 µm. (B) Quantification of viral binding from the image series in (A). Pooled data of three individual experiments (three biological replicates per experiment). n denotes the number of cells analysed. Experiments are color-coded; each dot represents one image, and each rectangle represents the experiment mean. Mean ± SD. ns, not significant (Two-way ANOVA). (C) Immunoblot of endogenous IGF1R and nucleolin in A549 WT and Arp2 KO cells. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) served as loading control. (D) Schematic of using PALM in combination with TIRF to assess IGF1R diffusion and clustering. This image was created using Biorender (License number: CT29AQJ52X). (E) Diffusion coefficient of A549 WT and Arp2 KO cells stably expressing IGF1R-mEos3.2. The data were obtained from sptPALM imaging. ns, not significant (unpaired two-tailed Student’s t-test). (F) Representative sptPALM reconstruction with tracks of individual IGF1R-mEos3.2 particles generated from 10,000 frames (frame interval 30 ms). Trajectories are color-coded. (G) Comparison of the IGF1R cluster size of A549 WT and Arp2 KO cells stably expressing IGF1R-mEos3.2. ns, not significant (unpaired two-tailed Student’s t-test). (H) Representative PALM image showing the surface distribution of IGF1R-mEos3.2 in an A549 WT cell.

Article Snippet: The antibodies used in this study were as follows: rabbit recombinant monoclonal anti-Arp2 antibody (1:2,000; EPR7979, ab129018, Abcam); mouse monoclonal anti-HSC70 antibody (1:5,000; sc-7298, Santa Cruz); GAPDH (D16H11) rabbit monoclonal antibody (1:1,000, 5174S, Cell Signaling Technology); IGF1R polyclonal antibody (1:500, BS-0227R, Bioss); nucleolin monoclonal antibody (1:500, 39-6400, Invitrogen); anti-Beta lactamase antibody (1:500, ab12251, Abcam); anti-SARS-CoV-2 nucleocapsid protein antibody (1:500, ABK79-E11-M, Abcalis); Palivizumab (10 μg/mL, a gift from Dr. Thomas Pietschmann, TWINCORE, Germany); HRP-conjugated goat anti-rabbit antibody (1:10,000; G-21234, Invitrogen); HRP-conjugated goat anti-mouse antibody (1:10,000; G-21040, Invitrogen); goat anti-Human cross-adsorbed secondary antibody, Alexa FluorTM 488 (1:200, A-11013, Invitrogen); goat anti-human IgG cross-adsorbed secondary antibody, HRP (1:10,000; A18811, Invitrogen).

Techniques: Confocal Microscopy, Incubation, Virus, Infection, Staining, Microscopy, Binding Assay, Western Blot, Control, Diffusion-based Assay, Stable Transfection, Expressing, Imaging, Two Tailed Test, Generated, Comparison

Journal: Advanced Science

Article Title: Transfer RNA‐Derived Small RNA‐5’tiRNAGln Drives HNRNPC Phase Separation to Promote Breast Cancer Bone Metastasis by Regulating IGF1R

doi: 10.1002/advs.77974

Figure Lengend Snippet: 5’tiRNA‐Gln could facilitate HNRNPC binding to IGF1R and enhance the mRNA stability. A, B: Depletion of HNRNPC represses activation of MEK/ERK signaling; C: Sequence motif analysis revealed an UUUACUA consensus enriched in HNRNPC‐bound RNA fragments; D: Genome browser tracks (hg19) showing HNRNPC eCLIP peaks (lower track) relative to size‐matched (SM) input (upper track) on the IGF1R transcript; E: Schematic representation of wild‐type (WT) and mutant (MUT) binding motifs within the coding sequence (CDS) region of IGF1R mRNA. The predicted HNRNPC‐binding consensus sequence (red) was mutated to generate the MUT reporter construct; F: Relative luciferase activity in cells co‐transfected with WT or MUT IGF1R reporter plasmids together with empty vector or HNRNPC overexpression plasmid (OE_HNRNPC) (n = 3 independent experiments; mean ± SD; ***p < 0.001); G: RIP‐qPCR revealed that HNRNPC binds to the mRNA of IGF1R, and repression of 5’tiRNA‐Gln reduced this binding efficiency in 231BO cells; H: Overexpression of HNRNPC variants showed that WT and MUT‐IDR1 enhanced the binding of HNRNPC to IGF1R, whereas MUT‐IDR2 had no such effect. Fusion of HNRNPA1 to MUT‐IDR2 restored the binding‐promoting effect in 231 cells; I: Remaining IGF1R mRNA after actinomycin D treatment. Data represent mean ± SD; n = 3 independent experiments, one‐way ANOVA with multiple comparisons test; J–K: Relative IGF1R mRNA expression levels in 231BO and 231 cells following HNRNPC knockdown (K) or overexpression of HNRNPC WT and its mutants (J); L: Western blot of IGF1R upon overexpression of 5’tiRNA‐Gln or treated with 1,6‐HD in 231 and BT‐549 cells. 1,6‐HD: 1,6‐hexanediol; M: Western blot of ERK signaling pathway‐associated proteins in breast cancer cells upon overexpression of 5’tiRNA‐Gln or knockdown HNRNPC; N: Western blot analysis of IGF1R and p‐ERK expression following HNRNPC knockdown and subsequent re‐expression (rescue) in 231BO and 231 cells.; O: Schematic illustration depicting the molecular mechanism of the 5’tiRNA‐Gln/HNRNPC/IGF1R axis in mediating breast cancer bone metastasis via phase separation. Data are shown as means ± SD. p value was calculated by one‐way ANOVA test with multiple comparisons (E, F, G, H, I).

Article Snippet: Other antibodies used included HNRNPC (Proteintech, 11760‐1‐AP), IGF1R (Proteintech, 20254‐1‐AP), β‐Actin (Proteintech, 20536‐1‐AP), HRP‐conjugated affiniPure goat antirabbit IgG (Proteintech, SA00001‐2).

Techniques: Binding Assay, Stability, Activation Assay, Sequencing, Analysis, Mutagenesis, Construct, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Over Expression, Expressing, Knockdown, Western Blot

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: The Journal of Biological Chemistry

Article Title: Downregulation of ClC-3 chloride channels in dorsal root ganglia neurons contributes to bone metastasis-induced pain

doi: 10.1016/j.jbc.2026.111268

Figure Lengend Snippet: Knockdown of Igf1r in DRG neurons disrupts intrathecal IGF1-induced activation of AKT signaling as well as the upregulated expression of HDAC2, the decreased expression of ClC-3 protein and pain hypersensitivity in na9;ve rats . A and B , RT-qPCR analysis and western blotting analysis of IGF1R mRNA and protein abundance in L4/5 DRG tissues from sham and BCP rats at 14 days after tumor cells inoculation. n = 6–7 rats per group. C , representative images showing the colocalization of IGF1R, HDAC2, and ClC-3 in DRG neurons. The scale bar represents 25 μm. D – F , western blotting analysis of p-AKT ( E ) and AKT ( F ) protein abundance in ipsilateral L4/5 DRG tissues obtained from IGF1-treated naïve rats that received intrathecal LV-shIGF1R and the control LV-GFP, respectively (n = 4 rats per group). D , representative blots are shown. G – I , western blotting analysis of HDAC2 ( H ) and ClC-3 ( I ) protein abundance in ipsilateral L4/5 DRG tissues obtained from IGF1-treated naïve rats that received intrathecal LV-shIGF1R and the control LV-GFP, respectively (n = 4 rats per group). G , representative blots are shown. J and K , assessment of ipsilateral PWT and PWL for IGF1-treated naïve rats that received intrathecal LV-shIGF1R and the control LV-GFP (n = 10 rats per group). L , assessment of animal’s locomotor function before and after intrathecal drug administration (n = 10 rats per group). Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗∗ p < 0.001, ns, not significant, unpaired t test for ( A )–( B ); one-way ANOVA followed by Dunnett's post hoc test for ( E )–( I ) two-way ANOVA with Sidak’s post hoc test for ( J )–( L ). BCP, bone cancer pain; DRG, dorsal root ganglion; HDAC2, histone deacetylase 2; IGF1, insulin-like growth factor 1; LV-GFP, lentivirus expressing green fluorescent protein; p-AKT, phosphorylated AKT; PWL, paw withdrawal latency; PWT, paw withdrawal threshold; RT-qPCR, real-time quantitative PCR.

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature and then incubated with the following primary antibodies at 4 °C overnight: rabbit anti-ClC-3 antibody (1:1000, Abcam), rabbit anti-HDAC2 antibody (1:1000, Merck Millipore), rabbit anti-IGF1 antibody (1:1000, Abcam), rabbit anti-IGF1R antibody (1:1000, Cell Signaling Technology),rabbit anti-p-AKT antibody (1:1000, Cell Signaling Technology), rabbit anti-AKT antibody (1:2000, Cell Signaling Technology), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology).

Techniques: Knockdown, Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Quantitative Proteomics, Control, Histone Deacetylase Assay, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of ClC-3 chloride channels in dorsal root ganglia neurons contributes to bone metastasis-induced pain

doi: 10.1016/j.jbc.2026.111268

Figure Lengend Snippet: Knockdown of Igf1r in DRG neurons impairs HDAC2-mediated transcriptional repression of Clc-3 gene, reduces neuronal excitability and attenuates pain hypersensitivity in bone metastasis model rats . A – E , western blotting analysis of p-AKT ( B ), AKT ( C ), HDAC2 ( D ) and ClC-3 ( E ) abundance in ipsilateral L4/5 DRG tissues from intrathecal LV-GFP and LV-shIGF1R treated rats at 14 days after tumor cells inoculation. n = 4 to 6 rats per group. A , representative blots are shown. F – H , electrophysiological analyses of neuronal excitability in ipsilateral L4/5 DRG neurons of bone metastasis model rats that received intrathecal LV-GFP and LV-shIGF1R, recorded at 14 days after tumor cells inoculation. F , representative neuronal action potentials evoked by a large depolarizing current pulse (1-s, 2-fold AP rheobase) are shown. The scale bar represents 20 mV, 100 ms. G and H , plots of threshold potential and rheobase are shown. n = 10∼20 cells from three rats per group. I and J , assessment of ipsilateral PWT ( I ) and PWL ( J ) from intrathecal LV-GFP and LV-shIGF1R treated rats after tumor cells inoculation. n = 10 rats per group. K , assessment of animal’s locomotor function by inclined-plate test, compared before and after drug administration. n = 10 rats per group. Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗∗ p < 0.001, ns, not significant, unpaired t test for ( B )–( H ); two-way ANOVA with Sidak’s post hoc test for ( I )–( K ). DRG, dorsal root ganglion; HDAC2, histone deacetylase 2; LV-GFP, lentivirus expressing green fluorescent protein; p-AKT, phosphorylated AKT; PWL, paw withdrawal latency; PWT, paw withdrawal threshold.

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature and then incubated with the following primary antibodies at 4 °C overnight: rabbit anti-ClC-3 antibody (1:1000, Abcam), rabbit anti-HDAC2 antibody (1:1000, Merck Millipore), rabbit anti-IGF1 antibody (1:1000, Abcam), rabbit anti-IGF1R antibody (1:1000, Cell Signaling Technology),rabbit anti-p-AKT antibody (1:1000, Cell Signaling Technology), rabbit anti-AKT antibody (1:2000, Cell Signaling Technology), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology).

Techniques: Knockdown, Western Blot, Histone Deacetylase Assay, Expressing