Anti rabbit igg hrp w401b

SW480 cells were seeded in 6-well plates at a density of 3.0 x 10⁵ cells per well and then treated on the next day with 20 µM SPOPP-3 (1), 20 µM SPOPP-5 (2), or 2% DMSO for 24 h. Cell lysates were prepared as previously described^{41 (link)}. For immunoblot analysis, protein samples were resolved on a 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The phospho H3 antibody (Ser10) (D2C8, 1:1,000, Cell Signaling) was used with an overnight incubation at 4 °C. Anti-GAPDH (G8795, clone GAPDH-71.1, 1:20,000, Sigma) was also used. Anti-mouse IgM-HRP (sc-2064, 1:4000, Santa Cruz Biotechnology), anti-mouse IgG-HRP (W402B, 1:4000, Promega) and anti-rabbit IgG-HRP (W401B, 1:4000, Promega) were used as secondary antibodies. All blots were visualized with the FluorChem Q system (ProteinSimple). Densitometry analysis was performed using the AlphaView Q software (ProteinSimple).

Cell lysis was performed with 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonic (CHAPS) acid buffer. Western blot analysis was performed using primary antibodies: polyclonal rabbit anti-APOL1 (HPA018885, 1:500, Sigma, St. Louis, MO), mouse monoclonal anti-GAPDH (CB1001, 1:10,000, Darmstadt, Germany), and secondary antibodies anti-rabbit IgG HRP (W401B, 1:10,000; Promega, Madison, WI) and anti-mouse IgG HRP (W402B, 1:10,000; Promega, Madison, WI).

The primary antibodies used were: anti-GAPDH (sc-47724, Santa Cruz), anti-AR (sc-7305, Santa Cruz), anti-DNMT3A (sc-365769, Santa Cruz), anti-EZH2 (612667, BD Transduction Laboratory), anti-DNMT(5032S, Cell Signaling Technologies), anti-EED (85322S, Cell Signaling Technologies), anti-SUZ12 (3737S, Cell Signaling Technologies), anti-Aurora A (14475T, Cell Signaling Technologies), antiH3K27me3 (9733S, Cell Signaling Technologies), and anti-PLK1 (B290751, BioLegend).
Tumor tissues (25–30 mg) or cellular pellets were lysates with RIPA buffer supplemented with cocktail phosphatase inhibitors (4906845001, Roche) and proteases inhibitors (5892953001, Roche). Protein concentration was determined by BCA reagent (A52255, Thermo Fisher Scientific); 30–50 μg of whole protein lysate was separated on 8–12% SDS–polyacrylamide gels and transferred onto PVDF membrane (88518, Thermo Fisher Scientific). The membranes were blocked with 5% milk in Tris-buffered saline with Tween-20 (TBST) for 30 min at RT, incubated overnight at 4 °C with primary antibodies, and incubated for 1 h at RT with secondary antibodies (anti-rabbit IgG HRP W401B and anti-mouse IgG HRP, W402B, Promega). The protein bands were visualized using the western bright quantum reagent (K-12042-D20, Advansta) and quantified using the Fusion Solo IV LBR system.