Hdac6 h 300

Cells were plated on coverslips and following overnight incubation were either fixed or transfected and incubated for 24 h. Cells were fixed with 3% paraformaldehyde, permeabilized in 0.5% Triton-X100 for 10 min and pre-blocked in 5% FBS diluted in PBS. Coverslips were incubated overnight with primary antibodies (see below), washed in PBS and incubated with secondary antibodies: anti-rabbit Alexa Fluor 488, anti-goat Alexa Fluor 488, anti-mouse Alexa Fluor 488, anti-mouse Alexa Fluor 647 and anti-rabbit Alexa Fluor 647. Cells were mounted with ProLong Gold antifade with DAPI (Invitrogen). Primary antibodies used in immunofluorescence: HDAC6 (H-300, Santa Cruz, Santa Cruz, CA, USA), α-tubulin (T5168, Sigma–Aldrich), RanBPM (K12, Santa Cruz, Santa Cruz, CA, USA), HA (HA-7, Sigma–Aldrich), α-tubulin (ab15246, Abcam), MAEA (ab151304, Abcam) and Twa (ab97653, Abcam). Confocal images were acquired using an inverted IX51 Olympus microscope equipped with a Perkin Elmer Spinning Disk confocal attachment with a 60× objective using Velocity software (Improvision). Z-stack image deconvolution was done using AutoQuant (Media Cybernetics, Rockville, MD, USA) software and image analyses for both plane and Z-stacks images were done using Imaris software (Bitplane, Zurich, Switzerland). Colocalization analyses were done using Imaris software using the top 2% of colocalized voxels.

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).