Horseradish peroxidase conjugated secondary antibody of goat anti rabbit igg h l

Total proteins were isolated from ESCs by RIPA buffer (Beyotime, Shanghai, China) and quantified with a BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins (20 µg) were separated by 10% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Roche, Mannheim, Germany). The membranes were blocked with 5% defatted milk and incubated with the primary antibodies as follows: anti-E-cadherin (ab40772, 1:10,000), anti-N-cadherin (ab76011, 1:5,000), anti-GAPDH (ab9485, 1:2,500), anti-MAP3K2 (ab33918, 1:10,000), anti-p-Erk1/2 (ab223500, 1:400), anti-Erk1/2 (ab184699, 1:10,000), anti-p-JNK (ab124956, 1:5,000), anti-JNK (ab199380, 1:2,500), anti-p-p38 (ab178867, 1:1,000), and anti-p38 (ab170099, 1:5,000) (all from Abcam, Cambridge, MA, USA) at 4°C overnight, followed by incubation with the horseradish peroxidase-conjugated secondary antibody of goat anti-rabbit IgG H&L (Abcam, ab175781, 1:10,000) at room temperature for 2 h. The proteins were visualized using an ECL kit (Cwbiotech, Beijing, China) and quantified with the Amersham Imager 600 (GE Healthcare Life Sciences, Little Chalfont, UK).

Total proteins were isolated from ESCs and uterine tissues using RIPA buffer (Beyotime, Shanghai, China) [34 (link)]. After quantification with a BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and separation by polyacrylamide gel electrophoresis, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Invitrogen) which were subsequently blocked with 5% defatted milk. After that, the membranes were incubated at 4°C overnight with the following primary antibodies: anti-SOCS3 (ab280884, 1:1000), anti-β-actin (ab115777, 1:200), anti-p-JAK2 (ab32101, 1:1000), anti-JAK2 (ab108596, 1:5000), anti-p-STAT3 (ab267373, 1:1000), anti-STAT3 (ab68153, 1:1000) (all from Abcam) followed by being incubated with the horseradish peroxidase-conjugated secondary antibody of goat anti-rabbit IgG H&L (Abcam, ab6702, 1:1000) at room temperature for 2 h. The proteins were visualized using an ECL kit (Cwbiotech, Beijing, China) and quantified with the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).