Anti β tubulin

Immunoblotting was performed as previously described [9 (link)]. In brief, whole protein lysates were separated by SDS/PAGE and transferred to nitrocellulose membranes (Whatman, Dassel, Germany). Membranes were incubated with the following primary antibodies diluted in 5% Milk/TBST-containing blocking solution overnight: anti-KPNA2 (rabbit polyclonal, 1:2000; abcam, Cambridge, UK), anti-stathmin (rabbit monoclonal, 1:1000; abcam), anti-E2F1 (rabbit polyclonal, 1:200; Santa Cruz, Heidelberg, Germany), anti-TFDP1 (rabbit polyclonal, 1:500; abcam), anti-ATF-2 (rabbit polyclonal, 1:200; Santa Cruz), anti-FBP-1/2 (goat polyclonal, 1:200; Santa Cruz), anti-c-JUN (rabbit monoclonal, 1:2000; Cell Signaling Technology, Frankfurt, Germany), anti-HMOX1 (rabbit monoclonal, 1:10,000; abcam), anti-GTSF1 (goat polyclonal, 1:200; Santa Cruz), anti-PARP (rabbit polyclonal, 1:500; Cell Signaling Technology), anti-β-tubulin (mouse monoclonal, 1:1000; Becton, Dickinson and Company, Franklin Lakes, USA) and anti-β-actin (mouse monoclonal, 1:10,000; MP Biomedicals, Illkirch, France). Blots were incubated with fluorescence-conjugated secondary antibodies (LI-COR Bioscience, Bad Homburg, Germany) for one hour and detection was performed using the Odysee Sa Infrared Imaging System (LI-COR Bioscience).

Spinal cord slices were collected in lysis buffer, homogenated, and quantified by BCA assay. Equal amounts of protein (20 μg/well) were resolved in SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked with 6% non-fat dry milk in phosphate buffered saline (PBS) for 1 h at room temperature (RT) and incubated overnight with the corresponding primary antibody diluted in blocking buffer (SMN, 1:500, BD Biosciences and anti-β-tubulin, Becton-Dickinson). After several washes, membranes were incubated for 1 h with an appropriate secondary antibody. Blots were developed using a chemoluminiscent mix and exposed to enzymatic chemoluminiscence (ECL) films (Amersham Pharmacia Biotech). Densitometry was carried out using ImageJ software.

Immunoblotting were executed as described previously.^{47 (link)} Simply, cells were washed with PBS and lysed in RIPA buffer (Invitrogen) added protease inhibitor (Sigma). Protein concentration of each sample was calculate by BCA protein assay kit (Thermo Fisher Scientific) to equal protein loading. Equivalent protein were underwent to SDS-PAGE, shifted to polyvinylidene fluoride membrane, interdicted in 5% skim milk for about 2 h at about 25 °C, then checked with relative primary antibodies. The following antibodies were used for analysis: anti-E-cadherin, anti-N-cadherin, anti-Vimentin and anti-SOX2 (Abcam plc, Cambridge, UK), anti-Nanog (Cell Signaling, Beverly, MA, USA), anti-β-actin (Sigma) and anti-β-tubulin (BD Biosciences, CA, USA). β-actin and β-tubulin was served as loading controls. Horseradish Peroxidase (HRP) Secondary Antibodies (Abcam) and an ECL Chemiluminescence Detection Kit (Thermo Fisher Scientific) were used to test combined antibody.