Dylight 680 conjugate

Immunoblotting was performed as previously described [26 (link)] using antibodies against GSR (N-Term) antibody (1 : 1000; Antikoerper-online.de, ABIN406391) and anti-actin antibody (1 : 4000; Millipore MAB1501). Secondary antibodies were anti-rabbit IgG (H + L) (DyLight™ 800 Conjugate) and anti-mouse IgG (H + L) (DyLight™ 680 Conjugate) from Cell Signaling Technology™ (1 : 30000).

Protein was extracted from ∼30 mg snap‐frozen tissue as previously described (Mallinson et al. 2012). Total and phosphorylated 4EBP1 (Ser⁶⁵) and p70S6k (Thr³⁸⁹) (Cell Signaling, Danvers, MA, USA) and PDK4 (Millipore, UK) were analysed with anti‐mouse IgG (H+L) (DyLight 800 Conjugate, Cell Signaling) or anti‐rabbit IgG (H+L) (DyLight 680 Conjugate, Cell Signaling) as the secondary antibodies. Blots were scanned and bands identified using the Odyssey Infrared Imaging System (LI‐COR Biosciences, Lincoln, NE, USA). Dual probing was used for total and phosphorylated proteins. Density volume was adjusted by subtracting the local background and then normalised with β‐actin (Sigma Aldrich, Dorset, UK).

After protein was extracted from renal tissues and cells, protein concentration was determined using the BCA method. After denaturation, 30–80 μg protein was separated using 12% SDS-PAGE. Proteins were transferred to PVDF membranes using wet transfer. The membranes were blocked in 5% skim milk at room temperature for 1.5 h, and then incubated overnight at 4°C with the following primary antibodies specific for: IL-1β (1:1000, #12242, Cell Signaling, Shanghai, China); IL-18 (1:1000, ab71495, Abcam, Shanghai, China); caspase-1 (1:500, GTX14367, GeneTex, United States); GSDMD (1:500, orb390052, Biorbyt, Cambridge, United Kingdom); and β-actin (1:5000, AC004, ABclonal, Wuhai, China). The membranes were then washed and incubated at room temperature in the dark for 2 h with the following secondary antibodies: anti-mouse IgG (H + L, DyLight^TM 800 Conjugate, 1:15000, #5257, Cell Signaling, Shanghai, China) and anti-rabbit IgG (H + L, DyLight^TM 680 Conjugate, 1:15000, #5366, Cell Signaling, Shanghai, China). A two-color infrared fluorescence imaging system was used to visualize protein bands. Protein concentration in the bands was quantified using ImageJ and normalized against that of β-actin, used as loading control.