Antiserum hrp tagged goat igg anti rabbit

Total protein lysates were isolated from the indicated cells using RIPA lysis buffer (JRDUN, Shanghai, China) with an EDTA-free protease inhibitor cocktail (Roche, Germany). An Enhanced BCA protein assay kit (Thermo Fisher, USA) was utilised to measure the protein concentration. A total of 25 μg protein was separated using a 10% SDS-PAGE gel and was transferred to a nitrocellulose membrane (Millipore, USA) overnight. The membranes were probed at 4 °C overnight with the primary antibodies: FOXP3 (1:1000), STAT5 (1:500), p-STAT5 (1:500) (Abcam, UK), and GAPDH (1:2000) (CST, USA) followed by incubation with the secondary antibody (antiserum-HRP tagged goat IgG anti-rabbit 1:1000; Beyotime, China) for 1 h at 37 °C. An enhanced chemiluminescence system (Tanon, China) was used to quantify the protein content. Each sample was tested in triplicate and GAPDH was used as the internal loading control.

RIPA lysis buffer (JRDUN, Shanghai, China) was used to extract protein as indicated. An enhanced BCA protein assay kit (Thermo Fisher, USA) was utilized to estimate the protein content. Total protein (25 μg) was fractionated by using 10% SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, USA) for 2 hours, which were probed at 4°C for 12 hours with the primary antibodies followed by incubation for 1 h at 37°C with the secondary antibody (antiserum-HRP tagged goat IgG anti-rabbit; 1 : 1000; Beyotime, China). An enhanced chemiluminescence system (Tanon, China) was utilized to quantify the content of protein expression. Each analysis was detected in triplicate. GAPDH was treated as the internal reference. Detailed information of the primary antibodies is provided in Supplementary Table 2.