The total protein of GAS was extracted and quantified using a BCA protein quantification kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were loaded into 15 wells (20 μg total protein per well) of 4–12% Bis-Tris gradient gels (NP0336BOX, Invitrogen, Waltham, MA, USA). After separation by electrophoresis, proteins were transferred to nitro-cellulose Regular Stack (IB23001, Invitrogen). Membranes were blocked with Odyssey blocking buffer (LI-COR), and then incubated with anti-NLRP3 (CST, 15101S), anti-ASC (Novus Biologicals, NBP1-78977, Centennial, CO, USA), anti-Caspase-1 (CST, 2225S), anti-GSDMDC1 (Santa Cruz, sc-393656, Santa Cruz, CA, USA), anti-MuRF1 (Santa Cruz, sc-398608) and anti-Atrogin1 (Abcam, ab168372) antibodies overnight at 4 °C. Then, after being washed four times (8 min each) by Tris-buffered saline (TBS) containing Tween-20 (TBST), the membranes were incubated with goat anti-rabbit or anti-mouse IgG secondary antibodies (LI-COR, 926-68071/926-32210, Lincoln, NE, USA) at 25 °C for 1 h. Next, the membranes were washed with TBST four times (8 min each) and then TBS twice (5 min each). The protein bands were detected by a near-infrared spectroscopy detection system (Odyssey CLX, LI-COR). All bands were analyzed semi-quantitatively using Image Studio Ver 5.2 software [58 (link)].
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