total igf 1r  (Cell Signaling Technology Inc)

Journal: Biomedicines

Article Title: Regulation of Soluble E-Cadherin Signaling in Non-Small-Cell Lung Cancer Cells by Nicotine, BDNF, and β-Adrenergic Receptor Ligands

doi: 10.3390/biomedicines11092555

Figure Lengend Snippet: Immunodepletion of sE-cad inhibited activation of EGFR and IGF-1R in A549 cells. Cells were incubated in serum-free media for 12 h, then treated for 72 h with 300 μL of media (0.5 μg/μL) immunodepleted using either hIgG (ID hIgG) or sE-cad (ID sE-cad) (Methods) prepared from cells either untreated (Control) or treated with BDNF (5 nM), nicotine (Nic, 1 µM), or epinephrine (Epi, 100 nM), as described in the legend. The phospho/total EGFR assay ( A , B ) and the phospho/total IGF-1R assay ( C , D ) were then carried out (Methods). Data were expressed as fold change relative to ID hIgG control untreated cells using the GraphPad 9.5.1 software (n = 5). Asterisks indicate a statistically significant difference between each treatment ID from sE-cad relative to the same treatment ID using hIgG, ** p < 0.0l. Please note the differences in scale. Asterisks indicate a statistically significant difference from the corresponding control for each cell line, using the Mann–Whitney test. For comparing three or more groups, the Kruskal–Wallis test, followed by Dunn’s multiple comparison test, was performed. The absence of asterisks indicates no significance; ** p < 0.0l.

Article Snippet: The Phospho-IGF-1R (Tyr1165/1166) and Total IGF-1R ELISA kits (RayBiotech, Peachtree Corners, GA, USA) were used to quantitate activated (phosphorylated) IGF-1R according to the manufacturer’s instructions and as previously reported [ ].

Techniques: Activation Assay, Incubation, Software, MANN-WHITNEY, Comparison

Journal: Biomedicines

Article Title: Regulation of Soluble E-Cadherin Signaling in Non-Small-Cell Lung Cancer Cells by Nicotine, BDNF, and β-Adrenergic Receptor Ligands

doi: 10.3390/biomedicines11092555

Figure Lengend Snippet: Summary of the findings from this study using A549 cells. The levels of MMP9 increased upon cell treatment with nicotine, epinephrine (Epi), or BDNF. Increased MMP9 levels lead to an increase in the levels of sE-cad in the media, increasing activation of EGFR and IGF-1R, enhancing PI3K and ERK1/2 signaling, and inhibiting p53 activity, resulting in decreased apoptosis and increased cell survival.

Article Snippet: The Phospho-IGF-1R (Tyr1165/1166) and Total IGF-1R ELISA kits (RayBiotech, Peachtree Corners, GA, USA) were used to quantitate activated (phosphorylated) IGF-1R according to the manufacturer’s instructions and as previously reported [ ].

Techniques: Activation Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Placental Remote Control of Fetal Metabolism: Trophoblast mTOR Signaling Regulates Liver IGFBP-1 Phosphorylation and IGF-1 Bioavailability

doi: 10.3390/ijms24087273

Figure Lengend Snippet: Inhibition of IGF-1R autophosphorylation in P6 cells using CM from HepG2 Cells treated with CM from PHT cells with RAPTOR and RICTOR siRNA. The ability of IGFBP-1 secreted from HepG2 cells treated with CM from PHT cells with the control, RAPTOR, and RICTOR siRNA to inhibit IGF-1 receptor autophosphorylation in P6 cells. ( A ) An equal amount of P6 cell lysate protein (25 µg) was loaded on 1-D gels for a Western blot using an antibody against p-IGF-1Rβ Tyr1135 . ( B ) Aliquots (1 mL) of HepG2 CM was used to treat a confluent culture of P6 cells in the absence of IGF-1 as a negative control while a serum-free sample containing 10 ng IGF-1 was a positive control. An equal protein amount (25 µg) of treated P6 cell lysate was loaded on 1-D gels for a Western blot using an anti-p-IGF-1R β antibody. The membranes were then stripped and re-probed with a total IGF-1Rβ antibody. The data were represented normalized to the control with Mean + SEM, analyzed with a one-way ANOVA, and corrected with a Bonferroni multiple comparison test. Means without a common letter differ are considered significant, p < 0.05.

Article Snippet: The membrane was stripped (Pierce Chemical Co) and re-probed with an antibody for total anti-IGF-1R (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Inhibition, Western Blot, Negative Control, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Cisplatin Resistance in Lung Cancer Cells by Nicotine, BDNF, and a β-Adrenergic Receptor Blocker

doi: 10.3390/ijms232112829

Figure Lengend Snippet: Phospho/Total IGF-1R ratio increased by cell treatment with epinephrine or nicotine and decreased by cell treatment with propranolol, while blocking IGF-1R activation by PPP increased cell sensitivity to cisplatin. ( A ) Cells (0.2 × 10 5 ) were grown in 10% FBS-supplemented media for 24 h. The following day, the cell monolayers were incubated in serum-free media for 24 h (Control), then incubated without or with FBS for 72 h. The phospho/total IGF-1R assay ( A ) was carried out on the same amount of protein (25 µL of 400 µg/mL total protein) of the cell lysate as described in the Methods section. The phospho/total IGF-1R assay was also carried out on A549 and H1299 cells grown as above then incubated in serum-free media for 72 h in the absence or presence of epinephrine (Epi, 100 nM), propranolol (Prop, 1 µM), nicotine (Nic, 1 µM), BDNF (5 nM), PPP (5 µM) or in combination without ( B , C ) or with cisplatin ( D , E ). Cell viability ( F ) and apoptosis ( G ) were determined in the presence of PPP (5 µM) without or with 10 µM cisplatin when using A549 cells and 30 µM cisplatin when using H1299 cells, as described in the Methods section. Data from five independent assays, each carried out in triplicate, were averaged, normalized, and expressed as fold change relative to untreated control cells (Control) using the GraphPad 9.4.1 software. The graphs summarize the results expressed as means ± SD (n = 5). Asterisks (*) indicate a statistically significant difference from the corresponding control for each cell line, Mann–Whitney test, while the absence of asterisks indicates no significance. * p < 0.05, ** p < 0.01.

Article Snippet: Activated (phosphorylated) IGF-1R was quantitated using the Phospho-IGF-1R (Tyr1165/1166) and Total IGF-1R ELISA kit (RayBiotech, Norcross, GA, USA), according to the manufacturer’s instructions.

Techniques: Blocking Assay, Activation Assay, Incubation, Software, MANN-WHITNEY

Journal: Toxicology research and application

Article Title: Differential Receptor Tyrosine Kinase Phosphorylation in the Uterus of Rats Following Developmental Exposure to Tetrabromobisphenol A

doi: 10.1177/23978473211047164

Figure Lengend Snippet: RTKs Overexpressed at PND33

Article Snippet: 20 Briefly, western blots were incubated overnight with the following specific primary antibodies diluted as indicated: 1:1000 for total and phosphorylated MAPKp44/42 tyrosine 204 (Cell Signaling Technology, Cat# 9102 and 9101); 1:1000 for total and phosphorylated EGFR tyrosine 845 (Cell Signaling Technology, cat# 2232 and 2231); 1:1000 for total and phosphorylated ErbB2 (Her2) tyrosine 1221/1222 (Cell Signaling Technology, Cat# 4290 and 2243); 1:1000 for total IGF-1R and phosphorylated IGF-1R tyrosine 1131/1146 (Cell Signaling Technology, Cat# 3027 and 3021); and 1:500 for HPRT (Santa Cruz Bio- technology, cat# sc-20975).

Techniques: Cell Differentiation, Transformation Assay, Migration

Journal: Toxicology research and application

Article Title: Differential Receptor Tyrosine Kinase Phosphorylation in the Uterus of Rats Following Developmental Exposure to Tetrabromobisphenol A

doi: 10.1177/23978473211047164

Figure Lengend Snippet: Protein expression of phospho-EGFR, phospho-ERBB2 and phospho-IGF1R after treatment with 0, 0.1, 25 and 250 mg/kg/day TBBPA at PND 90. (A) Western blot analysis of phospho-EGFR. The ratio of Phospho-EGFR to total EGFR was increased at all doses, but only significantly (*P<0.05) increased at 25 and 250 mg/kg/day TBBPA compared to controls. (B) Western blot analysis of phospho-ERBB2. The ratio of Phosho-ERBB2 to total ERBB2 was significantly (*P≤0.05) increased at all dose groups compared to controls. (C) Western blot analysis of phospho-IGF1R. The ratio of Phosho-IGF-1R to total IGF1R was significantly (*P<0.05) increased at all dose groups compared to controls. Experiments were repeated independently in triplicate.

Article Snippet: 20 Briefly, western blots were incubated overnight with the following specific primary antibodies diluted as indicated: 1:1000 for total and phosphorylated MAPKp44/42 tyrosine 204 (Cell Signaling Technology, Cat# 9102 and 9101); 1:1000 for total and phosphorylated EGFR tyrosine 845 (Cell Signaling Technology, cat# 2232 and 2231); 1:1000 for total and phosphorylated ErbB2 (Her2) tyrosine 1221/1222 (Cell Signaling Technology, Cat# 4290 and 2243); 1:1000 for total IGF-1R and phosphorylated IGF-1R tyrosine 1131/1146 (Cell Signaling Technology, Cat# 3027 and 3021); and 1:500 for HPRT (Santa Cruz Bio- technology, cat# sc-20975).

Techniques: Expressing, Western Blot