2.9. Western Blot Analysis STAT3/pSTAT3

Western blot analyses were performed to detect pSTAT3, STAT3, and α-Tubulin expression in iPSC-derived macrophages after IL-10 (20 ng/mL) stimulation. Thirty minutes after stimulation, cells were washed and detached. Cell pellets were resuspended in lysis buffer (50 mM HEPES, 150 mM NaCl, 50 mM NaF, 10 mM Na₄P₂O₇, 10% glycerin, 1% Triton X-100) with 1 µL of Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific). Protein lysate was loaded on a polyacrylamide gel, and gel electrophoresis was performed. Samples were blotted for 1.5 h at 4 °C (400 mA) onto nitrocellulose membranes. Membranes were blocked for 1 h at RT with 5% bovine serum albumin (BSA; PAA, Pasching, Austria) for pSTAT3 or 3% milk powder in PBS (Carl Roth, Karlsruhe, Germany) for STAT3 and α-Tubulin staining. STAT3 (1:2000, Cell Signaling Technology, Frankfurt, Germany), pSTAT3 (1:1000, Cell Signaling Technology, Frankfurt am Main, Germany), or α-Tubulin (1:10,000, Abcam, Cambridge, UK) antibody staining was performed overnight at 4 °C. Secondary antibody staining (Goat-anti-mouse 1:5000 or goat-anti-rabbit 1:5000; Abcam, Cambridge, UK) was performed according to the manufacturer’s description for 1 h at room temperature. Proteins were detected with the SuperSignal West Pico Chemoluminescent Substrate (Thermo Fisher Scientific, Darmstadt, Germany) with a FusionFX instrument (Peqlab, Darmstadt, Germany).