Horseradish peroxidase conjugated sheep anti rabbit igg

Total protein was extracted from L02, SMMC-7721, and Huh-7 cells. Western blotting was performed to detect the target proteins. The primary antibodies, including rabbit anti-human total protein kinase B (PKB/Akt), phospho (p)-Akt (Ser473), total extracellular signal-regulated protein kinases 1/2 (ERK1/2), p-ERK1/2 (Thr202/Tyr204), total epidermal growth factor receptor (EGFR), p-EGFR (Tyr1086), phosphatase and tensin homolog deleted on chromosome ten (PTEN), matrix metalloproteinase 2 (MMP-2), and beta-actin (β-actin) were purchased from Cell Signaling Technology (Danvers, MA, USA); rabbit anti-human B cell lymphoma/lewkmia-2 (Bcl-2), Bcl-2-associated X protein (BAX), CSE, CBS, and 3-MST antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The secondary antibody, horseradish peroxidase-conjugated sheep anti-rabbit IgG was purchased from Cell Signaling Technology (Danvers, MA, USA). The bands were semi-quantified with Image J software.

Western blotting was performed using an enhanced chemiluminescence system. Immunoblots were obtained for total protein extracts of the brains of 7-month-old wild-type (N = 3) and heterozygous Ubap1^+/E176Efx23 knock-in mice (N = 3). Briefly, the proteins were harvested from brain tissue by homogenization in RIPA buffer with a protease inhibitor cocktail (Roche). Equal amounts of proteins were separated on 10% SDS–polyacrylamide gels and then electrotransferred onto polyvinylidene difluoride membranes. After blocking with 3% BSA in PBS, the membranes were incubated with a primary antibody. After washing with PBS-0.1% Tween 20, filters were probed with horseradish peroxidase-conjugated sheep anti-rabbit IgG or rabbit anti-mouse IgG (Cell Signaling; Beverly, MA, USA). Immunoreactivity was detected with an enhanced chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK). The chemiluminescent signals were captured with a Fujifilm luminescent image LAS-4000 analyzer (Fujifilm, Tokyo, Japan). Two kinds of rabbit polyclonal anti-UBAP1 antibodies (Sigma-Aldrich, SAB1307218 and Invitrogen, PA5-49644) were used in this study.