Ha 11 clone 16b12 monoclonal antibody

All steps were performed at 4°C. Cell pellets of PC3-AR with R1881 (2 nM, 17 h) and RBN-2397 (100 nM, 17 h) treatments were re-suspended in the cell extraction buffer [20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5% Triton X-100, 1 mM PMSF, 2 mM DTT, 5 mM EDTA, 5 μg/ml each of aprotinin/leupeptin/pepstatin with 0.5 μM Veliparib], and then end-over-end rotated for 20 min. The extracts were clarified with centrifugation (16,800 × g) for 20 min, and then subjected to anti-Flag M2 magnetic beads (Sigma M8823–5ML) or anti-HA magnetic beads (Pierce 88837) binding for 3–4 h. The beads were collected by magnetic fields, washed five times with the wash buffer [20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1% Triton X-100, 2 mM DTT, 0.1 mM EDTA, 1 μg/ml each of aprotinin/leupeptin/pepstatin, 0.5 μM Veliparib), and then re-suspended in SDS-loading buffer followed with Western blot analyses. Immunoblots were detected on an Oddysey CLx instrument (LI-COR) using the following reagents: anti-HA (HA.11 Clone 16B12 Monoclonal antibody, BioLegend 901514, used at 1:1,000), Alexa Fluor 680 donkey anti-rabbit IgG(H+L) (Invitrogen A10043) and goat anti-mouse IgG(H&L) DyLight 800 conjugated (Rockland 618-145-002).

All steps were performed at 4°C. Cell pellets of PC3‐AR with R1881 (2 nM, 17 h) and RBN‐2397 (100 nM, 17 h) treatments were re‐suspended in the cell extraction buffer (20 mM Tris–HCl [pH 7.5], 100 mM NaCl, 0.5% Triton X‐100, 1 mM PMSF, 2 mM DTT, 5 mM EDTA, 5 μg/ml each of aprotinin/leupeptin/pepstatin with 0.5 μM Veliparib), and then end‐over‐end rotated for 20 min. The extracts were clarified with centrifugation (16,800×g) for 20 min, and then subjected to anti‐Flag M2 magnetic beads (Sigma M8823‐5ML) or anti‐HA magnetic beads (Pierce 88,837) binding for 3–4 h. The beads were collected by magnetic fields, washed five times with the wash buffer (20 mM Tris–HCl [pH 7.5], 100 mM NaCl, 0.1% Triton X‐100, 2 mM DTT, 0.1 mM EDTA, 1 μg/ml each of aprotinin/leupeptin/pepstatin, 0.5 μM Veliparib), and then re‐suspended in SDS‐loading buffer followed with Western blot analyses. Immunoblots were detected on an Oddysey CLx instrument (LI‐COR) using the following reagents: anti‐HA (HA.11 Clone 16B12 Monoclonal antibody, BioLegend 901,514, used at 1:1000), Alexa Fluor 680 donkey anti‐rabbit IgG(H + L) (Invitrogen A10043) and goat anti‐mouse IgG(H&L) DyLight 800 conjugated (Rockland 618‐145‐002).