His tag

Homogenized lung tissue or cell lysates were supplemented with phosphatase and protease inhibitors (both from Gold Biotechnology, Olivette, MO). Western blotting was performed as we previously described^{26 (link)}. Briefly, 8–16% polyacrylamide gels (Invitrogen Corp, Carlsbad, CA) were used to separate the proteins. We used GAPDH (Abcam, Cambridge, MA), 4-HNE (Percipio Biosciences, Burlingame, CA), Cys106-oxidized DJ-1 (HCA024, Bio-Rad, Hercules, CA), β-actin (Sigma, St. Louis, MO), lamin-B1, Tom 20, DJ-1, His-tag, VDAC1, IKBα, COX IV and PDI (all were purchased from Santa Cruz Biotechnology, Dallas, TX). Horseradish peroxidase (HRP)-conjugated AffiniPure donkey IgG were obtained from Jackson ImmunoResearch (West Grove, PA). The blots were then developed using an enhanced chemiluminescence western blotting kit according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Piscataway, NJ). Images were quantitated using ImageJ software.

Antibodies specific to p53 (DO-1 and FL393), His-tag and Myc-tag were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody specific to HA-tag (HA.11) was from Convance (Berkeley, CA) and those for α-tubulin and β-actin were from Sigma (St. Louis, MO). Polyclonal RBEL1 antibody was generated in our laboratory [15 (link)-16 (link)]. Proteasome inhibitor MG132 was purchased from Sigma (St. Louis, MO), glutaraldehyde (GA) and bovine serum albumin (BSA) were from Fisher Scientific Inc (Pittsburg, PA).

Antibodies for hnRNPK, His tag, N-WASP and cortactin were obtained from Santa Cruz (Santa-Cruz, CA). Oregon-green gelatin was purchased from Sigma Aldrich. Phalloidin-TRITC was obtained from Invitrogen (Carlsbad, CA).

MCF7 and T47D were purchased from ATCC. HEK293FT was purchased from Life Technologies. Tamoxifen-resistant MCF7 clones were generated by dose-escalation 4OHT-treatment in phenol red-free RPMI1640 culture medium supplied with 10% charcoal-stripped FBS (Life Technologies): 200 nM for 2 weeks, 500 nM for 2 weeks and then 2 μM for 4 weeks. Single clones were picked up under microscope and then expanded in 2 μM 4OHT-containing phenol red-free RPMI1640 culture medium supplied with 10% charcoal-stripped FBS. Antibodies used in this report were: COPS5 (A300-014A, Bethyl Laboratories), NCoR (PA1-844A, Thermo Scientific) for western blot (WB) and IHC, NCoR (sc-1609, Santa Cruz) for IP and ChIP, ERα (SC-543, Santa Cruz) for WB, ERα (61035, Active Motif) for ChIP, GPS1 (ab4535, Abcam), CSN2 (PA1-24686, Thermo Scientific), CSN6 (sc-47965, Santa Cruz), NEDD8 (Y297) (ab81264, Abcam), PCAF (sc-13124, Santa Cruz), HDAC1 (sc-6298, Santa Cruz), HDAC3 (sc-17795, Santa Cruz), HA-tag (MMS-101P, Covance), His-tag (sc-803, Santa Cruz), actin (A-3853, Sigma), tubulin (T6074, Sigma) and secondary antibodies were purchased from Sigma. Compounds used were Tamoxifen (T5648, Sigma), 4OHT (94873, Sigma), E2 (E8875, Sigma), fulvestrant (I4409, Sigma), flavopiridol (S2679, Selleckchem) and MG132 (S2619, Selleckchem).

Exosomes (10 μg or 10⁹ particles) were resuspended in PBS with protease inhibitors. The suspension was mixed with Laemmli buffer, heated at 95 °C for 5 min, and chilled on ice for 5 min before loading onto SDS gel. Immunoblots probed with antibodies for proteins: Tsg-101 (C-2, 1:1000, Santa Cruz), CFP (C192, 1:1000, ATCC), 19kDa-lipoprotein (IT-19, 1:1000, ATCC), His-tag (1:500, Santa Cruz), CD81 (1:500, SBI), CD63 (1:500, Systems Bioscience), and, DsRed (632393, 1:1000, Clontech). Primary antibody incubation was followed with HRP-conjugated secondary antibodies (1:25,000, Pierce) and detected using enhanced chemiluminecence kit (Pierce). As loading controls, primary mouse mAbs against either alpha-Tubulin, 1/1000 dilution (Cat. T9026, Sigma) or Lamp-1 1/500 dilution (SC-17768, Santa Cruz Biotechnology or 1D4B, DHSB) were used.