β actin mouse monoclonal antibody

For hippocampus and cortex homogenate, rats were anaesthetized and transcardially perfused only with saline. Brains were carefully removed and dissected to separate hippocampus and cortex, which were then split with RIPA Lysis Buffer. Protein concentration of homogenate was measured by BCA Protein Assay Kit (Beyotime) to prepare samples at the same concentration for Western blot (10% SDS-PAGE, 40 μg total proteins). Targeted strips were incubated with primary antibodies (anti-NF-κB p65 antibody, ab16502, Abcam; anti-NF-κB pp65 antibody, ab86299, Abcam; β-actin mouse monoclonal antibody, Beyotime) and then secondary antibodies (biotin-labeled goat anti-rabbit IgG, Beyotime; biotin-labeled goat anti-mouse IgG, Beyotime), followed by visualization with Immobilon Western HRP Substrate (Merck, Darmstadt, Germany). IL-1β and TNF-α in the homogenate were analyzed by ELISA (Rat IL-1β/IL-1F2 DuoSet ELISA, DY501, R&D, Minneapolis, MN, USA; rat TNF-α DuoSet ELISA, DY510, R&D) according to kit instructions.

Each protein sample, i.e. teratocytes culture medium, cell‐free hemolymph of parasitized and non‐parasitized P. xylostella was mixed with SDS protein loading buffer (Sangon, China) and boiled in water for 10 min. Afterwards, the protein samples were separated by 12% SDS‐PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio‐Rad, USA) using a Trans‐Blot SD Cell and System (Bio‐Rad, USA) at 16 V for 20 min. The PVDF membrane was blocked with 3% BSA overnight and washed in 1 × TBST buffer. The CvT‐serpin15 in protein samples were detected with CvT‐serpin15 polyclonal antibodies (1: 200) as primary antibodies and the β‐actin as an internal standard was detected with β‐actin mouse monoclonal antibody (1: 2000) (Abcam, UK) followed with anti‐rabbit/mouse secondary antibodies conjugated with IgG‐horseradish peroxidase (HRP) (ABclonal Tech., China). Protein bands on PDVF membranes were detected using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, USA) and photographed by ChemiDoc MP Imaging System (Bio‐Rad, USA).