Goat anti rabbit igg antibody

Apoptosis proteins Bax, Bcl-2, and cleaved caspase 3 were determined after 72 h, using rabbit antibodies (Cell Signal. Technol., Danvers, MA, USA). The secondary antibody was a goat-anti-rabbit-IgG antibody (ZSGB-BIO, Beijing, China).
Ferroptosis modulins transferrin (TF), ferroportin (FPN), and glutathione peroxidase 4 (GPX4) and the autophagy protein microtubule-associated protein light chain 3 (LC3) were detected after 48 h. Antibodies against TF, FPN, and GPX4 were from Abcam (Cambridge, UK), and the anti-LC3 antibody was from Cell Signal. Technol. The secondary antibody was a goat-anti-rabbit-IgG antibody (Abcam).
β-Actin was the reference, using a rabbit antibody (Cell Signal. Technol.). Bands were assayed with the software Image Lab (Bio-Rad Lab., Hercules, CA, USA), and the level of a target protein was quantified with the density ratio.

The cell lysates from each sample was subjected to electrophoresis and transferred onto a polyvinylidene difluoride membrane. The membrane was probed with primary antibodies and HRP-conjugated secondary antibodies, and then developed using enhanced chemiluminescence (ECL) system (Beyotime Biotechnology, China). The density of the bands was quantified by densitometry using GelPro 3.2 software. The β-actin protein served as internal control to normalize the expression level of other proteins.
The primary antibodies included an anti-RAC1 antibody (Catalog number 8631, 1:1000 dilution, Cell Signaling Technology, USA), an anti-A20 antibody, an anti-FAK Phospho (pY397) antibody, an anti-FAK Phospho (py861) antibody, an anti-FAK antibody, an anti-E-Cadherin antibody and an anti-N-Cadherin antibody (Catalog number 2644–1, 2211-S, 2153–1, 1700-S, 1702–1 and 2447–1, 1:1000 dilution, Epitomics, USA), an anti-RIP1 antibody (Catalog number ab125072, 1:1000 dilution, Abcam, China), and an anti-β-actin antibody (Catalog number PR-0255, 1:2000 dilution, ZSGB-BIO, China). The secondary antibodies included a goat anti-rabbit IgG antibody and a goat anti-mouse IgG antibody (Catalog number ZB-2301 and ZB-2305, 1:2000 dilution, ZSGB-BIO, China).

Celastrol (purity of > 99%, cat. No. HY-13067) and PI3K activator (purity of > 99%, cat. No. HY-151527) were purchased from MedChem Express (MCE, shanghai, China) and was dissolved in dimethyl sulfoxide (DMSO) to generate a stock concentration of 10 nM, stored at − 80 °C.p-PI3K (cat. No. 4228), PI3K (cat. No. 4257), p-AKT (cat. No. 4060), AKT (cat. No. 4691), p-mTOR (cat. No. 5536), mTOR (cat. No. 2983), and β-actin (cat. No. 4967) were purchased from Cell Signaling Technology (CST, Woburn, USA). rabbit anti-mouse IgG antibody, and the secondary antibody, goat anti-rabbit IgG antibody was purchased from ZSGB-BIO (Beijing, China). BCA protein assay kit and Cytoplasmic Protein Extraction Kits were purchased from Beyotime (Jiangsu, China).