α tubulin clone b 5 1 2 t5168

Proteins were extracted from cell cultures using RIPA (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and concentrations measured using the Micro BCA Protein Assay Kit (Thermo Fisher Scientific, Reinach, Switzerland) on a Multiplate Reader Synergy HT (Bio-Tek, Luzern, Switzerland) with the KC4 software. The following antibodies were used: HA-Tag (6E2) Mouse mAb #2367 (Cell Signaling, LabForce AG, Nunningen, Switzerland), GFP N-terminal G1544 (Sigma, Buchs, Switzerland), PARP (46D11) Rabbit mAb #9532 (Cell Signaling, Labforce AG, Nunningen, Switzerland), p62/SQSTM1 P0067 (Sigma, Buchs, Switzerland), Ub (A-5) sc-166553 (Santa Cruz Biotechnology, LabForce AG, Nunningen, Switzerland) and α-Tubulin Clone B-5-1-2 T5168 (Sigma, Buchs, Switzerland).

SLUG (sc-166476), Tyrosinase (sc-20035), SOX9 (sc-20095), Fra-1 (clone D-3, sc-376148), nucleolin C23 (sc-8031) and ZEB1 (sc-25388) (Santa Cruz Biotechnology, INC, Dallas, TX, USA), Snail (#3895) and p27Kip1 (#2552) (Cell Signaling Technology, Leiden, Netherlands), Microphthalmia (Ab-1, #OP126L, Calbiochem Thermo Fisher, Waltham, MA, USA), Twist (clone 2C1a #ab50887 AbCam, Cambridge, UK), PRAME (TA309818, OriGene, Rockville, MD, USA), ZEB2 (HPA003456, Atlas Antibodies) and SPARC (OSN4.2, #M124 from Takara, Kusatsu, Japan) were used in accordance to the manufacturer’s instructions. A mouse monoclonal and a rabbit monoclonal Abs were utilized against E-Cadherin (clone 36 BD #610181, Transduction Laboratories and clone 24E10, Cell Signaling Technology, Leiden, Netherlands #3195). A specific polyclonal rabbit antibody was generated against a human SCD5 synthetic peptide (aa 313-327) (Eurogentec Group, Liege, Belgium). β-actin (Clone AC-15 #A5441) and α-Tubulin (clone B-5-1-2 #T5168 (Sigma Aldrich St. Louis, MO, USA) were used as loading controls.

Antibodies against neurofilament-light chain Ab9035 (Western blot), and NFL mouse monoclonal, Cat# MCA-DA2 (immunostaining), tyrosinated α-tubulin (clone YL1/2; ab6160) and stathmin-1 (clone EP1573Y; ab52630) were obtained from Abcam. The specificity of these antibodies and in particular the stathmin antibody has been tested in previous studies [5 (link), 66 (link)]. After stathmin-1 knockout [5 (link)] or lentiviral knockdown [66 (link)] the corresponding Stathmin-1 band was completely abolished in Western blots. Neurofilament-heavy chain antibody (AB5539) was obtained from Millipore, eIF2α (D7D3), p-STAT3^Y705 (D3A7), and Stat3 (124H6) (9139S) antibodies from Cell Signaling Technology. γ-tubulin (clone GTU-88), tau (T-6402), acetylated-α-tubulin (clone 6-11b-1; T7451) and α-tubulin (clone B-5-1-2; T5168) antibodies were purchased from Sigma-Aldrich. The Stathmin 2 (SCG10) antibody was a kind gift from the lab of Dr. Gabriele Grenningloh, EPFL [15 (link)].