Igf1 r antibody

IGF1-R antibody was purchased from Abcam (ab182408, Cambridge, UK). β-Actin antibody was purchased from Thermo Scientific (Rockford, IL, USA). LC3-I, LC3-II, p62, ATG-7, ATG-12, BNIP3, and HIF1-α were purchased from Novus (Novus Biologicals, Centennial, CO, USA). Pro-caspase 3 was purchased from Cell Signaling Technology (Beverly, MA, USA). Pro- and cleaved caspase 9 was purchased from Abcam (Cambridge, UK).

Paraformaldehyde (4%) was used to fix cells for 20 min, and 0.5% Triton X-100 in PBS was utilized to permeabilize the cells for 10 min. Subsequently, 10% fetal bovine serum was used to block the cells for 1 h. Finally, the cells were incubated with IGF-1R antibody (Abcam, United States) at 4°C overnight. On the following day, FITC-labeled immunofluorescence secondary antibody (Bioworld, United States) was added to plates of cells for incubation for 1 h in the dark. Then, 4-6-diamino-2-phenindole (Bioworld, United States) was used to visualize the nucleus. Images were captured using a fluorescence microscope (Leica, Frankfurt, Germany).

Whole cell lysates from S2VP10L, MiaPaCa-2, S2013, and SCC-1 cells were collected to determine IGF1R expression. The cells were plated at a density of 5x10⁵ cells per well in a 6-well plate 24 h prior to protein harvest. Cells were then lysed in a solution containing 1 % NP-40, 1 % protease inhibitor, and 1 % phosphatase inhibitor (Thermo) in deionized water. The lysates were centrifuged at 13,000×g for 10 min. Total protein concentration was determined via Bradford assay (Bio-Rad, Hercules, CA, USA).
Approximately 50 µg of total protein was dissolved in deionized water, loading buffer, and reducing agent (Life Technologies, Carlsbad, CA, USA). Proteins were separated using NuPage 4–12 % Bis–Tris gel and transferred onto a nitrocellulose membrane by iBlot (Life Technologies). The membrane was blocked in blocking buffer (Li-Cor) for 30 min and then incubated overnight at 4 °C with IGF1R antibody (Abcam, Cambridge, England) at a concentration of 1 µg/mL and β-Actin antibody (Thermo) at a concentration of 1:3000. The membrane was then washed 3× with TBS (20 mM Tris–HCl, 150 mM NaCl in diH₂O) for 10 min each, incubated with donkey anti-mouse IRDye 680RD and donkey anti-rabbit IRDye 800CW secondary antibodies (Li-Cor) for 1 h, washed 3× with TBS for 10 min each, and scanned using Li-Cor Odyssey infrared scanner. Dosimetry was performed using Li-Cor software.