Goat anti rabbit igg hrp affinity purifiedpab

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).