In-cell Western assays for IGF-1R

Cells were seeded at 5 × 10⁴ cells per well in 96-well tissue culture plates in complete medium for 24 hours, washed three times with PBS, and serum-starved for 4 hours followed by stimulation with IGF-1 (10 ng/ml) for 10 min or left in serum-free medium or complete medium. Cells were immediately fixed with 100 μl of a freshly prepared fixing solution (4% formaldehyde/PHEM) and incubated at room temperature for 30 min followed by quenching with 50 mM ammonium chloride for 20 min and washing with PHEM. Cells in wells with complete medium or incubated with p-AKT/AKT and one 3X set of the secondary control antibody were permeabilized using 0.1% Triton/PHEM for 5 min and washed with PHEM. All wells were then blocked for 1 hour with 50 μl of blocking buffer (5% goat serum/PHEM). Primary antibody (IGF-1R IR3 for surface and Cell Signaling IGF-1Rβ catalog no. 3027 or p- Ser⁴⁷³/AKT) was added for 2 hours with gentle rocking followed by washing and incubation with secondary antibody. Goat anti-mouse IRDye 680 (1:1000) (Odyssey infrared imaging system, catalog no. 926-32220) or Goat anti-rabbit IRDye 800 (1:1000) (Odyssey infrared imaging system, catalog no. 926-32211) were used to detect primary antibodies. Syto60 (Invitrogen) was also included (1:10,000) as a cell-permeable nucleic acid stain that fluoresces at 680 nm and was detected using the Odyssey infrared scanner. Cells were washed five times with PHEM. Plates were dried, and IGF-1R (surface and total levels) intensity was quantified using the Image studio quantification software with normalization to Syto60 levels.