Goat anti mouse igg hrp

The total proteins from thymic samples were extracted as described previously^{19 (link)}. The protein samples (10 μg/lane) were separated on 12% SDS-PAGE gels, and then transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Blot analysis was performed with a mouse anti-NF-κB1 monoclonal antibody (Santa Cruz Biotechnology, Inc., sc-8414, 1:1000), a mouse anti-NF-κB2 monoclonal antibody (Santa Cruz Biotechnology, sc-7386, 1:1000), a mouse anti-RelA monoclonal antibody (Santa Cruz Biotechnology, sc-8008, 1:1000), a mouse anti-RelB monoclonal antibody (Santa Cruz Biotechnology, sc-166416, 1:1000), and a mouse anti-c-Rel monoclonal antibody (Santa Cruz Biotechnology, sc-6955, 1:1000) at 4 °C overnight, respectively. Then, the membranes were washed and incubated with goat anti-mouse IgG-HRP (horseradish peroxidase) secondary antibody (Biosharp, BL001A, 1:2000) for 1 h at room temperature. Chemiluminescent substrate was used according to the manufacturer’s instructions (Tiangen Biotech) to detect immunoreactive protein. An anti-GAPDH antibody (Santa Cruz Biotechnology, sc-20357, 1:1000) was applied as an internal control protein. The blots were exposed to X-ray film, and densitometry of autoradiograms was performed using Quantity One V452 (Bio-Rad Laboratories). Relative expression levels of target proteins were normalized using the GAPDH.

The fixed hepatic tissues were treated as described previously [26 (link)]. The anti-IκBβantibody (Santa Cruz Biotechnology, sc-390622, 1:200) and anti-IKKγantibody (Santa Cruz Biotechnology, sc-166398, 1:200) were used for immunohistochemical localization of IκBβ and IKKγ in the liver tissue. After being rinsed three times for 5 min, sections were incubated with goat anti-mouse IgG-HRP (Biosharp, Hefei, China, BL001A) in a 1:1000 dilution. The negative control was treated with goat anti-mouse IgG instead of IκBβ and IKKγ antibodies. A DAB kit (Tiangen Biotech Co., Ltd., Beijing, China) was used to visualize the antibody binding sites, and then the nucleus was stained with haematoxylin. Tissue sections were photographed using a light microscope (Nikon Eclipse E800, Tokyo, Japan) with a digital camera (AxioCam ERc 5s), and the intensity of staining was analysed through the photos by two investigators in a blinded fashion. The intensity of staining was scored on a scale of 0 to 3:0, no staining; 1, weak staining; 2, strong staining; 3, stronger staining, as described previously [24 (link)].

After 24 h of aloin, chloroquine, rapamycin and starvation administration, HOS and MG-63 cell lines were lysed in RIPA buffer with protease and phosphatase inhibitor cocktail. Protein samples of same amount were separated by western blot. Blot intensity was analyzed by Image J (v1.51). The primary antibodies applied in this research were as follows: AKT, ATG5, Beclin1, Bcl-2, bcl-xl, cleaved caspase 3, cleaved caspase 7, cleaved caspase 9, cleaved PARP, mTOR, pho-mTOR (SER2448), pho-AKT (SER473), PI3Kα (p110), SQSTM1 (P62), LC3B. Beta-actin was used as internal reference. The secondary antibodies were as follows: goat anti-rabbit IgG–HRP (1:2000, Biosharp) and goat anti-mouse IgG–HRP (1:2000, Biosharp).