antibodies against phospho irβ igf1rβ  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: Anthrax ET activates Rac1 and RTK signaling to induce F-actin reorganization and endothelial permeability

doi: 10.1016/j.isci.2025.113682

Figure Lengend Snippet: Early IGF1R activation by ET contributes to toxicity (A) IGF1R activation in HBMECs was assessed by monitoring the levels of IGF1R phosphorylated at tyrosine 1135 and 1136 relative to total IGF1R by western blot. IGF1R activation was observed following 250 ng/ml treatment with ET, ET mut or 500 ng/ml PA within 10 min of treatment. (B) HBMECs were treated with 250 ng/mL ET for 4 h, in the presence or absence of the IGF1R inhibitor AG1024. Whole-cell lysates were incubated with PAK-PBD beads. Bead bound Rac1-GTP and total Rac1 levels in cell lysate were determined by WB with anti-Rac1 antibody. IGF1R inhibition decreased Rac1-GTP levels in ET treated cells. (C) Quantification of whole-cell area of HBMECs treated with ET (250 ng/mL) for 4 h, in the presence or absence of the IGF1R inhibitor AG1024. Cells were fixed and stained with HCS CellMask Green to visualize the cytoplasm. Whole-cell area was quantified from 8 wells for each treatment (n = Total cell number) using automated image analysis software. IGF1R inhibition completely rescued ET-induced reduction in whole-cell area. Statistical significance was evaluated using an unpaired Student t test and represented using standard symbolism ( ∗∗∗∗ p < 0.0001). Error bars indicate standard deviation. (D) HBMECs were treated with 250 ng/mL ET for 4 h, in the presence or absence of the IGF1R inhibitor AG1024 and stained with Alexa Fluor 568-Phalloidin to visualize F-actin. AG1024 blocked ET-induced F-actin rearrangement. Scale bar represents 30 μm. (E) Activation of downstream IGF1R effector AKT was assessed using a phospho-specific antibody (anti-phospho-Ser-473 AKT) in HBMECs treated with 250 ng/mL ET for the indicated time periods. Total and phosphorylated AKT levels were determined by WB. ET induced transient phosphorylation of AKT within 30 min of treatment. (F) Activation of CFL1 and SSH1 was assessed by monitoring the levels of pCFL1 (Ser3) and pSSH1 (Ser978) in whole-cell lysates by WB. PI3K inhibitor GDC-0941 prevented CFL and SSH1 dephosphorylation induced by a 4 h ET treatment (250 ng/ml and 500 ng/ml). Double arrow indicates pSSH1 in the lower panel. (G) HBMECs were treated with 250 ng/mL ET for 4 h, in the presence or absence of the PI3K inhibitor GDC-0941, and stained with Alexa Fluor 568 conjugated phalloidin to visualize F-actin. GDC-0941 blocks ET-induced cortical actin rearrangement. Scale bar represents 30 μm. These experiments (A–G) were performed 2 times.

Article Snippet: Primary antibodies for pIGF1Rβ (#3024S), IGF1Rβ (#3027S), pEGFR (#2234S), EGFR (#4405S), pAKT (#4060S), AKT (#9272S), pERK (#9101S), ERK (#9102S), pCFL1 (#3311S) and CFL1 (#3318S), were all purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Activation Assay, Western Blot, Incubation, Inhibition, Staining, Software, Standard Deviation, Phospho-proteomics, De-Phosphorylation Assay

Journal: The Journal of Biological Chemistry

Article Title: Missense mutations of the ephrin receptor EPHA1 associated with Alzheimer’s disease disrupt receptor signaling functions

doi: 10.1016/j.jbc.2024.108099

Figure Lengend Snippet: The P460L mutation and impaired N-glycosylation decrease EPHA1 cell surface localization. A , proteins on the surface of HEK293 cells transiently expressing EPHA1 WT or the indicated mutants were biotinylated and pulled down with streptavidin beads. Pulled down proteins (cell surface) and cell lysates were probed as indicated. IGF1 receptor β (IGF1Rβ) was detected as a control that should have similar cell surface localization in all samples. The graph shows cell surface expression of the indicated mutants normalized to EPHA1 WT based on quantification of immunoblots. The bars show means and SE from quantification of five to seven experiments, with the individual data points shown as black dots. ∗∗∗, p < 0.001 for the comparison with WT using one-way ANOVA and Dunnett’s multiple comparisons test. B , cell surface biotinylation experiment similar to (A) comparing the indicated mutants. C , HEK293 cells stably expressing EPHA1 WT or the P460L mutant were treated with cycloheximide for the indicated times and probed by immunoblotting with the EPHA1 SAM antibody and a β-tubulin antibody as a loading control. The graph shows EPHA1 levels normalized to EPHA1 level at time 0 of cycloheximide treatment, quantified from the immunoblots from four experiments. The error bars represent SEs. ∗, p < 0.05 by unpaired Student’s t test for the comparison between EPHA1 WT and L460L at the 14 h time point.

Article Snippet: The blots were also probed with an anti-IGF1Rβ antibody (#3027, Cell Signaling Technology) as a transmembrane protein control and with an anti-GAPDH antibody (#2118S, Cell Signaling Technology) as a cytoplasmic protein control.

Techniques: Mutagenesis, Glycoproteomics, Expressing, Control, Western Blot, Comparison, Stable Transfection