Anti total tubulin antibody

After isolation, content determination and electrophoresis, proteins were elettroblotted [46 (link)] and incubated with a polyclonal rabbit anti-CRBP-1, anti-Creb1, anti-CD44, anti-c-Jun, anti-Nox4, anti-p53, anti-RXRα, anti-RARα (Santa Cruz Biotechnology), anti-RARβ, (Abcam), anti-phosphorylated v-akt murine Jesi AN, Italy) and then sequenced using PyroMark Q24 thymoma viral oncogene homolog (pAkt Ser⁴⁷³), anti-AKT (pan), anti-phosphorylated extracellular-signal-regulated kinases (pErk1/2), anti-phosphorylated epidermal growth factor receptor (anti-EGFR Thr669), anti-EGFR antibody (Cell Signaling Technology, Danvers, MA, USA), anti-keratin 5 (clone H-40, Santa Cruz Biotechnology), mouse anti-vimentin (clone J144, Abcam), anti-keratin 14 (LL001, Santa Cruz Biotechnology) and anti-total tubulin antibody (Sigma-Aldrich). Revelation and densitometric blot analysis were performed in three independent experiments and Akt and EGFR activity expressed as phospho/total protein ratio [47 (link)].

Gene expression analysis of HaCaT cells was performed using Signosis array (Signosis, Inc. Santa Clara, CA, USA). Briefly, RNA was extracted, reverse-transcribed into cDNA in the presence of biotin-dUTP and a profile of 24 genes for human AKT and NF-κB pathway cDNA plate array (Signosis) respectively, was carried out through the detection of streptavidin-HRP. Luminescence reported as relative light units (RLUs) on a microplate luminometer, according to the manufacturer’ instruction. Experiments performed in triplicate. For western blot analyses, after extraction, proteins were blotted onto nitrocellulose membranes and incubated with anti-phosphorylated v-akt murine thymoma viral oncogene homolog (pAkt Ser⁴⁷³), anti-AKT (pan), anti-phosphorylated extracellular-signal-regulated kinases (pErk1/2; Cell Signaling Technology, Danvers, MA, USA), anti-interleukin 6 (IL-6, R&D Systems, Minneapolis, USA), anti-interleukin 8 (IL-8, Abcam, Cambridge, UK) and anti-total tubulin antibody (Sigma-Aldrich). Revelation and densitometric blot analysis were performed in three independent experiments. Membranes were reblotted with anti-tubulin to ensure equal loading. Akt activity was expressed as phospho/total protein ratio. Results are mean values ± SEM of the three different experiments. ^*p < 0.05, ^**p < 0.005 and ^***p <0,001. Abbreviations: ADU, arbitrary densitometric units.