Veriblot ip detection reagent

WM1366 melanoma cells were harvested by trypsinization. Cells were resuspended in ice-cold co-IP buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1% Na-deoxycholate, 0.05% SDS, and protease inhibitor) and lysed by mild sonication (three 5-s pulses). The cell debris were removed by centrifugation, and the supernatant was subjected to extensive DNase I digestion. Co-IP was performed by incubating 500 µg of the lysate with 4 µg of anti-AR (5153S; CST) or anti-RNA pol II (ab26721; Abcam) antibodies in 500 µl co-IP buffer for 8 h at 4°C. A mock reaction was also performed by incubating rabbit IgG (as control) with the lysate. Prewashed Protein A magnetic beads were added, and the incubation was continued for another 1 h. The beads were washed three times with 1 ml of co-IP buffer, resuspended in SDS-PAGE sample loading buffer, and incubated for 20 min at 98°C. The samples were resolved on 8% SDS-PAGE, and immunoblotting was performed with anti-AR (06-680; Merck Millipore), anti-RNA pol II (ab26721; Abcam), and anti-Ku70 (GTX101820; GeneTex) antibodies. VeriBlot IP Detection Reagent (HRP; ab131366; Abcam) was used as secondary antibody (1:200 dilution) to selectively detect target protein bands, without interference from the denatured IgG heavy and light chains. Details of the antibodies used are provided in Table S6.

For immunoprecipitation of endogenous FAT1, HUVECs were lysed in immunoprecipitation (IP) buffer (150 mM NaCl, 25 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1% NP-40, 5% glycerol) supplemented with protease inhibitor cocktail (catalog #4693159001, Roche) on ice. Samples were centrifuged at 30,000 × g for 15 min. Supernatant was incubated with 2 µg of the anti-FAT1 antibody overnight at 4 °C on a rotating platform. Thereafter, A/G-Sepharose beads (catalog #sc-2003, Santa Cruz) pre-equilibrated in IP buffer were added and incubated for 1 h at 4 °C. A/G-Sepharose beads were then collected by centrifugation at 3500 × g for 1 min, and proteins were eluted with Laemmli buffer after being washed three times. Samples were subjected to SDS-PAGE using VeriBlot IP detection reagent (catalog #ab131366, abcam). For immunoprecipitation of heterologously expressed proteins, HEK-293 cells were transfected with eukaryotic expression plasmids carrying the indicated cDNAs for 24–36 h using Lipofectamine 2000 (catalog #11668019, Thermo Fisher Scientific) and were then processed as described above.

Cell lysates were prepared using RIPA Buffer (Boston BioProducts) supplemented with protease inhibitors (Sigma) and phosphatase inhibitor (Active Motif). For immuoprecipitation experiments, cell lysates were incubated with desired antibodies in Tris buffered saline buffer at 4 °C overnight, then protein G agarose (Beyotime Biotechnology) was added to each sample for another 2 h incubation, followed by five times wash with PBS solution and boiling for IB. To avoid the interference from denatured IgG heavy chain, VeriBlot IP Detection Reagent (ab131366, Abcam) was used for IP detection. The antibodies against ARV7 (ab198394), Bcl-2 (ab59348), MCL-1 (ab28147) and Bim (ab15184) were obtained from Abcam while antibodies against cleaved-PARP (5625), Bid (2002), UBE2C (14234), p-H3(ser10) (9701), BubR1 (4116), and Cdc20 (14866) were purchased from Cell Signaling Technology, and the antibodies against GFP (sc-9996), p53 (sc-126), Mad-2 (sc-47747), and ubiquitin (sc-8017) were obtained from Santa Cruz. Proteintech is the provider of antibodies against β-actin.