Acetyl α tubulin k40

Phosphorylated form of H2AX, α-tubulin acetylation, Ki-67 and Nanog expression were evaluated, by IHC on paraffin embedded sections cut from tumor xenografts, using the following antibodies: γH2AX(Ser139, Millipore), Ki-67 (MIB-1, Dako, Milan, Italy), Nanog (Cell Signaling), acetyl-α-tubulin (K40) (Sigma-Aldrich). For each tumor, three different 5μm paraffin sections were analyzed and examined by light microscopy. Sections were scanned at 200× magnification. H2AX and Ki-67 nuclear immunoreaction of tumor cells was counted in four high-power fields (400× magnification) per section. For each tumor, counts were averaged to determine the number of positive cells. Immunoreactions were revealed by a streptavidin-biotin enhanced immunoperoxidase technique in an automated autostainer (Bond^TM Max, Leica BioSystem). The immunohistochemical detection of apoptosis was performed by TUNEL assay using a commercial kit (In Situ Cell Death Detection Kit, POD, Roche). The assay was performed according to the manufacturer's instructions. For each tumor, three different 5μm frozen sections were analyzed and examined by light microscopy. Sections were scanned at 200× magnification. Apoptosis of tumor cells was counted in four high-power fields (400× magnification) per section. Evaluation of the IHC results was performed independently and in blinded manner by two investigators.

Western blot analysis followed published procedures^{70 (link)}. In brief, crude extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose membranes. Blocked filters were probed with antibodies against acetyl-α-tubulin K40 (1/10,000, Sigma-Aldrich, St. Louis, MO, USA, #T7451), α-tubulin (1/1000, Santa Cruz, sc-5286), phospho-AKT Ser473 (1/2000, Santa Cruz, sc-7985), pan-AKT (1/1500, Cell Signaling, #9272), HDAC6 (1/1000, Santa Cruz, sc-11420), EGFR (1/1000, Santa Cruz, sc-03), phospho-ERK1/2 Thr202/Tyr204 (1/2000, Cell Signaling, #9106), pan-ERK1/2 (1/2000, Cell Signaling, #4695), and actin (1/100,000, Chemicon, MAB1501). Signals for enhanced chemiluminescence were acquired with a Bio-Rad ChemiDoc™ MP imaging system and quantified.

Western blot analyses of total protein extracts were performed as previously described [12 (link)]. Immunodetection was performed using antibodies directed to: H3 histone (abcam), H3 acetylated histone (Millipore, Billerica, MA, USA), α-tubulin (DM1A) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), acetyl-α-tubulin (K40) (Sigma-Aldrich), γH2AX (Ser139) (Millipore), PARP cleaved (Millipore), PARP (Santa Cruz Biotechnology) β-actin (Sigma-Aldrich), HSP72/73 (Calbiochem, San Diego, CA, USA), anti-mouse or anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase conjugated antibodies (Cell Signaling; Amersham Biosciences, Freiburg, Germany). Antibody binding was visualized by enhanced chemiluminescence method (Amersham Biosciences) according to manufacturer's specification and recorded on autoradiographic film (Amersham Biosciences). Densitometric evaluation was performed using Image J software and normalized with relative controls depending on the Analysis.