Mouse anti ha 11 16b12

Samples were separated on 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride Immobilon-P membranes (Millipore). Membranes were blocked and incubated with primary antibodies overnight at 4°C, washed, and incubated for 2 h with horseradish peroxidase (HRP)-coupled secondary antibodies at room temperature. All antibodies were diluted in 5% nonfat milk in TBST (100 mM Tris-Cl [pH 7.4], 150 mM NaCl, 0.1% [vol/vol] Tween 20). Mouse anti-DnaK (8E2/2; Enzo), mouse anti-HA.11 (16B12; Covance), mouse anti-IκBα (L35A5; Cell Signaling), rabbit anti-IκBα phospho-S32 (14D4; Cell Signaling), rabbit anti-FLAG (Sigma), rabbit anti-GFP (Invitrogen), rabbit anti-arginine-GlcNAc (EPR18251; Abcam), rabbit anti-MLKL (Sigma), rabbit anti-MLKL phospho-S345 [EPR9515(2); Abcam], rabbit anti-TRIM32 (Abcam), rabbit anti-tubulin (EPR16774; Abcam), and rabbit anti-actin (Sigma) were used. The HRP-conjugated goat anti-mouse and anti-rabbit secondary antibodies were purchased from Dako. The immunoblots shown are representative of three independent experiments.

HEK293T, human prostate adenocarcinoma LNCaP, Du145 and PC3 cells lines were purchased from the American Type Culture Collection, Manassas, VA, USA. The Docetaxel resistant cell lines Du145 and PC3 were developed as previously described [31 (link)]. HEK293T were cultured in DMEM (BioWest, Nuaillé, France), Docetaxel sentitive and resistant Du145 cells and LNCaP cells were cultured in RPMI-1640 (BioWest) and PC3 DS and PC3 DR in F12K nutrient mixture medium (Thermo Fisher Scientific, Waltham, MA, USA). Culture media were supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U of penicillin/ml, 100 μg of streptomycin/ml, and 0.1 mM non-essential amino acids (all from BioWest). Docetaxel resistant cell lines were maintained with 2.5 nM of Docetaxel (Sigma-Aldrich, St. Louis, MO). Antibody to PTOV1 was produced and purified as previously described [8 (link), 9 (link)]. Additional antibodies were obtained from: goat anti-actin (I-19) (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-HA.11 (16B12) (Covance, MA); mouse anti-Cyclin B1(V152) (Abcam, Cambridge, MA); rabbit anti-PARP1 (H-250) (Santa Cruz Biotechnology). Lentiviral vectors carrying short-hairpin RNA (shRNA, TRCN0000143905, TRCN0000140104 and TRCN0000139737) to PTOV1 were obtained from Sigma.