Anti creb1

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)].

Samples were prepared according to the protocol of the SimpleChIP Plus Enzymatic Chromatin IP Kit (catalog no. 9005, Cell Signaling Technology). Immunoprecipitates were exposed to anti-CREB1 (catalog no. SC-240, Santa Cruz Biotechnology) and anti-H3K27Ac antibodies, plus Protein G magnetic beads. After reversing, DNA was enriched; this was followed by qPCR.

The polyclonal antibodies anti-ERK5 and anti-phospho ERK5-pT²¹⁸EY²²⁰ were from Cell Signaling Technology (Danvers, MA, USA). The anti-ERK5 antibody used in immunoprecipitation and immunofluorescence experiments (C-7) and the anti-Cdc37 and anti-CREB1 antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA). The anti-GST, anti-FLAG and anti-HA monoclonal antibodies were from Sigma-Aldrich (Saint Louis, MO, USA); the anti-Hsp90β from Merck Merck Millipore (Burlington, MASS, USA); and the anti-SUMO antibody from Invitrogen (Carlsbad, CA, USA). The anti-Ubc9 antibody was a gift of Dr. R. Hay (College of Life Sciences, University of Dundee, Dundee, UK).