Acetylated α tubulin 6 11b 1

The antibodies used in this study are the following: rabbit polyclonal: α-GAP45 (Plattner et al., 2008 (link)), α-IMC1 (Frénal et al., 2014 (link)), α-Cpn60 (Agrawal et al., 2009 (link)), α-HSP70 (Pino et al., 2010 (link)), α-ARO (Mueller et al., 2013 (link)), α-GAC, α-Centrin1 (Kerafast), α-Ty and α-Myc (gifts from Chris Tonkin, WEHI). Mouse monoclonal: α-ACT (Herm-Götz et al., 2002 (link)) α-ATrx (DeRocher et al., 2008 (link)), α-ISP1 (Beck et al., 2010 (link)) α-Ty (BB2), α-Myc (9E10), α-SAG1, α-MIC2, ROP2-4 (gifts from J-F Dubremetz, Montpellier), acetylated α-tubulin (6-11B-1; Santa Cruz Biotechnology). Rat α-HA (3F10, Roche). For immunofluorescence assays, the secondary antibodies Alexa Fluor 405-, Alexa Fluor 488-, Alexa Fluor 594-conjugated goat α-mouse, α-rabbit, or α-rat antibodies (Life Technologies) were used. For western blot analyses, secondary peroxidase conjugated goat α-rabbit or mouse antibodies (Sigma) were used.

Whole cell extracts were prepared as described [2 (link)] and resolved by SDS-PAGE (between 8% and 12%) and transferred to polyvinylidene difluoride (PVDF) membranes. Samples were analyzed with the following antibodies: HDAC6 (H-300, Santa Cruz, Santa Cruz, CA, USA, and PA5–11240, ThermoFischer Scientific), HA (HA-7, Sigma–Aldrich), Acetylated α-tubulin (6–11B-1, Santa Cruz, Santa Cruz, CA, USA), α-tubulin (T5168, Sigma–Aldrich), β-Actin (I-19, Santa Cruz, Santa Cruz, CA, USA), RanBPM (5 M, Bioacademia, Japan), Rmnd5A (NBP1–92337, Novus Biologicals) and muskelin (C-12, Santa Cruz, Santa Cruz, CA, USA). The blots were developed using Clarity ECL Western Blotting Substrate (BioRad, Hercules, CA). Quantifications were done using Image Lab (BioRad, Hercules, CA) and ImageJ software. Co-immunoprecipitation experiments were performed in 0.25% NP-40 and 100 mM KCl lysis buffer and were carried out overnight at 4 °C with antibodies to HA (HA-7, Sigma–Aldrich), OctA-Probe (D-8 Santa Cruz, Santa Cruz, CA, USA), HDAC6 (D-11 Santa Cruz, Santa Cruz, CA, USA) and RanBPM (F1 Santa Cruz, Santa Cruz, CA, USA). Immunoprecipitates were isolated with PureProteome Protein G Magnetic Beads (EMD Millipore, Billerica, Massachusetts) or Dynabeads Protein G (Invitrogen, Life Technologies, Burlington ON, Canada).

Dexamethasone (Dex)-sensitive MM.1S and Dex-resistant MM.1R were obtained from the American Type Culture Collection (Manassas, VA, USA). RPMI-8226 and U266, NCI-H929, SKO, and LP1 human MM cells were provided by Changzheng Hospital, Shanghai. All MM cell lines were cultured with RPMI-1640 + 10% FBS medium. BIS was synthesized in Shanghai Institute of Materia Medica. BIS was dissolved first in DMSO at a concentration of 10 mM, and then in culture medium (0.5–8 μM) immediately before use. SAHA was purchased from MedExpress company.
Bortezomib was obtained from Selleck Chemicals for the in vitro studies. It was dissolved first in DMSO at a concentration of 20 mM, and then in culture medium before use. Bortezomib formulated with mannitol was dissolved in saline for the in vivo studies.
Histone H3, acetyl-Histone H3 (Lys23), and poly(ADP-ribose) polymerase antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Acetylated-α-tubulin (6-11B-1) and alpha-tubulin antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).