Hrp labeled goat anti rabbit mouse igg h l

Protein lysates from cells were prepared in lysis buffer and centrifuged at 12000 rpm at 4 °C. The primary antibodies used were JMJD2C (Santa Cruz, USA), β-catenin (CST, USA), c-Myc (CST, USA), ITGBL1 (Abcam, USA), PCNA (CST, USA) and GAPDH (CST, USA). The secondary antibody used was HRP-labeled goat anti-rabbit/mouse IgG (H + L) (Beyotime, China). Each band was quantitatively analyzed using quantity one Software and normalized to the expression of GAPDH in the same lane.

HCT116/L-OHP cells in the logarithmic growth period were used to establish the control group and groups of different doses of ZJW. L-OHP was combined with different concentrations of ZJW (0, 10, 15, and 20 μg/mL) for 48 h. The protein was extracted and quantified by a BCA protein assay kit (Beyotime, China), and the protein samples were treated. A 10% SDS-PAGE gel, sample protein, and marker (Thermo, China) were prepared. The proteins were then transferred to PVDF membranes after electrophoresis and sealed at room temperature for 2 hours. The closed membrane was immersed in the primary antibody and incubated overnight at 4°C. The next day, TBST was used to wash the film three times, each time for 10 min, and the membrane was soaked in the secondary antibody and incubated at room temperature for 2 hours. After washing the film, it was colored with ECL chemiluminescence solution (Cytiva, China), and the gray value was analyzed by ImageJ software. The primary antibodies used were MDR1/ABCB1 (CST, USA), ABCG2 (CST, USA), MRP4/ABCC4 (CST, USA), JNK2 (CST, USA), phospho-SAPK/JNK (CST, USA), and GAPDH (CST, USA). The secondary antibody used was HRP-labeled goat anti-rabbit/mouse IgG (H + L) (Beyotime, China). Each band was quantitatively analyzed using ImageJ software and normalized to the expression of GAPDH in the same lane.