Goat anti mouse igg hrp affinity purified pab

Tissue was lysed in RIPA buffer (Beyotime) containing protein inhibitors (Roche). Protein concentration was determined by BCA protein assay (ShareBio). Proteins were mixed with SDS Page loading buffer (Beyotime) and incubated at 100 °C for 10 min. ILC2s (1 × 10⁵ cells) were lysed in 50 μl 1 × SDS loading buffer containing phosphatase inhibitors (Roche). Then, protein lysate per lane was run through Gels and transferred to NC Membrane (Merck Millipore). The membrane was blocked for 1 h in 5% nonfat dried milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated overnight with primary antibody at 4 °C. The membrane was then washed 3 times in TBST and incubated with an HRP-conjugated secondary antibody for 1 h at room temperature. Detection was performed with ECL western blotting detection reagents (ShareBio). The following antibodies were used: Rabbit anti-mouse CRH(Abcam,ab184238,1/5000); Rabbit anti-mouse POMC(Abcam, ab210605, 1/5000); Rabbit anti-mouse p-STAT3 (CST,9131s,1/1000); Rabbit anti-mouse p-STAT5 (CST,9359s,1/1000); Rabbit anti-mouse p-p65 (CST,3033s,1/2000); Beta Actin Monoclonal antibody (Proteintech, 66009-1-Ig,1/5000); Goat Anti-Rabbit IgG HRP Affinity Purified PAb (R&D Systems, HAF008,1/1000); Goat Anti-mouse IgG HRP Affinity Purified PAb (R&D Systems, HAF007,1/1000).

HAECs were lysed in RIPA buffer (Sigma-Aldrich) including protease inhibitors (Complete Protease Inhibitor Cocktail, Roche). Cell lysates were resolved on SDS–PAGE gels and then transferred to nitrocellulose membranes. Proteins were detected using primary antibodies against KLF4 (1:1,000, EPR3550(2)ABC, Abcam), KLF2 (1:75, ab139699, Abcam) and GAPDH (1:1,000, clone 6C5, Millipore), and horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1,000, Goat Anti-Mouse IgG HRP Affinity Purified PAb, R&D Systems; Goat Anti-Rabbit IgG HRP Affinity Purified PAb, Santa Cruz). Protein bands were visualized using an enhanced chemiluminescence detection system (ECL Advance, GE Healthcare Life Sciences) and an LAS 3000 Imager (Fuji Photo Film Co., Ltd.) and were quantified using Multigauge software (Fuji Photo Film). Intensities of the KLF4 and KLF2 bands were expressed as a percentage of those of the GAPDH bands.