Anti α tubulin

For the Multinucleation assay, cells were fixed and stained with anti-α-tubulin antibodies and Hoechst 33342 (Invitrogen). Then, cells were analyzed for the presence of more than one nucleus per cell. Cells connected by midbodies were considered multinucleated. The viability of cells was assayed using a cell counting kit-8 (Dojindo, Tokyo, Japan) according to the manufacturer’s instructions.

For cell‐based experiments, cells were washed three times in PBS and resuspended with lysis buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P‐40, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mM Na3VO4, and 1 mM phenylmethylsulfonylfluoride (PMSF), and 25 mM NAM and trichostatin A (TSA) for 20 min and centrifuged for 20 min at 13,800 g; the insoluble fraction was discarded. The lysates were fractionated by SDS/PAGE and transferred to nitrocellulose filter (NC) membranes. The membranes were blocked for 1 h with Tris‐buffered saline with Tween 20 (TBST) containing 5% (mass/vol) nonfat dry milk. After incubation with primary antibodies (anti‐acetylated tubulin (K40) (Proteintech), anti‐α‐tubulin (Invitrogen), anti‐SIRT2(Abcam) or anti‐His (Abmart), each diluted 1:1000), the membranes were washed and incubated with HRP‐conjugated anti‐mouse (Cell Signaling Technology) or anti‐rabbit secondary antibodies (Cell Signaling Technology), followed by detection using ECL Western blotting substrate (Bio‐Rad).

Anti-His-tag (mouse), anti-Rac (mouse), anti-E-cadherin (rabbit), anti-GAPDH (rabbit), anti-histone H3 (rabbit), anti-α-tubulin (rabbit), anti-rabbit IgG (goat), anti-mouse IgG conjugated with Alexa Fluor^® 488 (goat), anti-rabbit IgG conjugated with Alexa Fluor^® 594 (goat) were purchased from Invitrogen (Oregon, USA); anti-mouse IgG was obtained from Dako (rabbit, California, USA). GDP and a non-hydrolyzable GTP analogue, guanosine 5′-[β,γ-imido]triphosphate (GppNHp), were obtained from Jena Bioscience GmbH (Jena, Germany). TC100 insect cell media, fetal bovine serum, antibiotics (penicillin and streptomycin) and 10% Pluronic F-68 were obtained from PAN-Biotech GmbH (Aidenbach, Germany). Phosphatidylserine (PS), Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM), phosphatidylinositol 4,5-bisphosphate (PIP2), and Folch I and Folch III brain lipid extracts were purchased from Sigma-Aldrich (Munich, Germany). PIP₃ is from Merck (Darmstadt, Germany). All other standard reagents, including detergents (Table S1 in File S1) were obtained from Carl Roth GmbH (Karlsruhe, Germany) or Merck-Millipore (Darmstadt, Germany).

Sections were successively incubated at room temperature in a blocking solution [5% w/v milk powder protein/phosphate-buffered saline (PBS) and 0.05% v/v Tween solution] for 1 h and in solutions containing primary probes for 1 h. The dilutions of probes in 5% w/v milk/PBS/Tween solution were as follows: 1:200X dilution for Anti-α-Tubulin (Invitrogen), and 1:1000X dilution for the cellulose binding module CBM3a. Sections for Anti-α-Tubulin labeling were extensively washed (5 min, three times) then incubated in milk powder protein/PBS solution containing the FITC conjugated secondary antibody for 1 h followed by another extensive wash. Sections for CBM3a detection were incubated in 1:1000X anti-histidine antibody in buffer solution for 1 h. After an extensive wash, the sections were incubated in the presence of FITC conjugated secondary antibody for 1 h and washed again. Sections incubated without primary antibodies were used as negative controls. Wild type sections treated with lichenase (from Bacillus sp., Megazyme; 1:1000 dilutions) for 1 h prior to CBM3a labeling were also used as controls. Brightness and contrast adjustments were done using ImageJ (NIH, Bethesday, MD, USA², 1997–2014).