Anti human α tubulin antibody

Protein extracts were obtained by lysing cells in TNN buffer supplemented with 1× Complete ULTRA protease inhibitor (Roche, Basel, Switzerland) and 1× PhosSTOP (ThermoFisher Scientific, Waltham, MA, USA). A total of 25 µg protein was separated on 4–10% Criterion TGX Stain-Free Protein Gels (BioRad, Hercules, CA, USA) or Mini-PROTEAN TGX Stain-Free Protein Gels (BioRad) and immediately transferred to Trans-Blot Turbo Midi PVDF Transfer Packs (BioRad) or Trans-Blot Turbo Mini Transfer Packs (BioRad). The blots were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween-20 (TBST) for 1 h at RT followed by incubation with anti-human CDKN2A antibody (Abcam, ab108349, 1:1000), anti-human Mesothelin antibody (Abcam, ab93620, 1:150), anti-human Podoplanin antibody (Abcam, ab236529, 1:500), and anti-human NF2 antibody (Abcam, ab109244, 1:10,000) overnight at 4 °C. The detection antibody HRP anti-rabbit IgG (BioLegend, San Diego, CA, USA, 410406, 1:1000) was incubated for 1 h at RT. Membranes were imaged using either Clarity Western ECL Substrate (Biorad) or SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) on a Fusion FX (Vilber Lourmat, Eberhardzell Germany). GAPDH and α-Tubulin were used as loading controls, anti-human GAPDH antibody (ThermoFisher Scientific, MA5-15738, 1:1000), and anti-human α-Tubulin antibody (Sigma-Aldrich, T5168, 1:1000).

Cell lysates were prepared from implanted tumor tissues using a whole cell lysis buffer supplemented with Halt protease and phosphatase inhibitor cocktail (Mammalian Protein Extraction Reagent; Thermo Scientific, Rockford, IL, USA). Protein samples were processed using standard western immunoblotting procedures. Membranes were incubated overnight at 4°C with the following antibodies in Can Get Signal Immunoreaction Enhancer Solution 1 (Toyobo, Osaka, Japan): anti-human cleaved caspase-3 antibody (1∶500) (Cell Signaling Technology, Danvers, MA, USA), anti-human cleaved caspase-9 antibody (1∶500) (Cell Signaling Technology), anti-human poly-ADP-ribose polymerase-1 (PARP) antibody (1∶500) (Cell Signaling Technology), and anti-human α-tubulin antibody (1∶5000) (Sigma-Aldrich). After washing, the membranes were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) in Can Get Signal Immunoreaction Enhancer Solution 2, and exposed with ECL Prime Plus western blotting detection system reagent (GE Healthcare Bio-Sciences). The signals were detected using a Chemilumino analyzer LAS-3000 mini (Fujifilm, Tokyo, Japan) [9] .