Alpha tubulin dm1a

For western blot analysis, cells were washed three times with ice-cold PBS and lysed in RIPA buffer (137 mM NaCl, 20 mM Tris–HCl, pH 7.4 10% (v/v) glycerol, 1% (v/v) Triton X-100 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS) supplemented with PhosSTOP Phosphatase Inhibitor Cocktail tablets (Roche), cOmplete Protease Inhibitor Cocktail tablets (Roche) and Benzonase Endonuclease (Merck Millipore). Protein lysates were separated on 4–12% Bis-Tris PAA gels (Invitrogen, ThermoFisher Scientific) and transferred to nitrocellulose membranes. Membranes were blocked in 5% w/v non-fat milk in PBS plus 0.05% v/v Tween 20 (PBST) for an hour and then probed with primary antibodies for 1 h at room temperature or overnight at 4°C. After washing, membranes were incubated with secondary antibodies coupled to horseradish peroxidase (HRP) for an hour at room temperature, washed and proteins were detected using chemiluminescence with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher). Primary antibodies used for western blots were: KRas clone 2C1 (cat. C175665; Lifespan Bioscience); vinculin clone hVIN-1 (cat. V9131; Sigma); alpha-tubulin (DM1A, Cell Signalling Technology); Lamin B1 (Cell signalling technologies), secondary antibodies were sourced from Abcam (donkey anti-mouse- HRP, donkey anti-rabbit-HRP).

The following antibodies, from the indicated sources, were used: rabbit anti-phospho-STAT3 (#9145), rabbit anti-STAT3 (#8768), rabbit anti-phospho-JAK1 (#74129), rabbit anti-myc (#9145), alpha-tubulin (DM1A, #3873) all from Cell Signaling Technology; mouse anti HA11 (#901514) and anti-Tuj1 (both from Biolegend); mouse anti-myc 9E10 (UPenn Cell Center); rabbit anti-GFP #A11122 (Invitrogen); mouse anti-JAK1 #610231 (BD Transduction Labs), mouse-anti NeuN #MAB377 (Millipore Sigma), Calnexin #10286 (Abcam). Unless otherwise noted, all chemicals were from ThermoFisher Biosciences and were of the highest reagent grade available.

The cells were rinsed twice with ice-cold PBS and then lysed in a RIPA buffer containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The protein lysates were purified by centrifugation at 14,000 rpm at 4 °C for 15 min. The total protein and an appropriate weight marker (Chamelon^® Vue Pre-stained Protein Ladder, LI-COR Biosciences, Lincoln, NE, USA) were separated using SDS-PAGE and transferred to nitrocellulose membranes by applying a wet tank blot transfer system. Membranes were blocked with 5% skim milk for 60 min, following the incubation of the primary antibodies at 4 °C overnight. The incubation of the appropriate secondary antibodies was performed for 60 min at room temperature. Finally, the detection of the proteins was carried out by using the WesternSure PREMIUM Chemiluminescent Substrate and a C-DiGit Blot Scanner (LI-COR Biosciences).
The following antibodies were used: PTEN (138G6; Cell Signaling, Danvers, MA, USA, 1:1000), AKT (polyclonal; Cell Signaling, 1:1000), phospho-AKT (Ser473; D93; Cell Signaling, 1:1000), S6 ribosomal protein (5G10; Cell Signaling), phospho-S6 ribosomal protein (Ser240/244; D68F8; Cell Signaling; 1:1000), histone H3 (D1H2; Cell Signaling; 1:2000), alpha-tubulin (DM1A; Cell Signaling; 1:1000), anti-rabbit and anti-mouse IgG antibodies produced in goat (Sigma Aldrich, St. Louis, MO, USA).