Anti cdc37

Whole cell extracts (WCE) were obtained by lysis with 20 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA, 2 mM EGTA supplemented with 0,5% Triton X-100 (Sigma-Aldrich), protease inhibitor cocktail (Sigma-Aldrich), phosphatase inhibitor cocktail (Thermo Scientific), 1 mM phenyl-methyl-sulfonyl fluoride (PMSF; Sigma-Aldrich), 1 μM okadaic acid (Sigma-Aldrich), dithiothreitol (DTT; Fluka). Twenty to 50 μg of WCE were subjected to SDS-PAGE, transferred to PVDF membranes and immunoblotted with the following primary antibodies: anti-CK2α (kindly provided by Dr S. Sarno, University of Padua, Italy); anti-CDC37 (Santa Cruz Biotechnology, Santa Cruz, CA) anti-phospho-Ser13 CDC37 (Abcam); anti-CK2β (BD Biosciences, USA); anti-phospho-Ser209 CK2β (Assay Biotech) and anti-GAPDH (Ambion); anti-RelA (Abcam) and anti-phospho-Ser529 RelA (Santa Cruz Biotechnology, Santa Cruz, CA). As secondary antibodies: anti-rabbit IgG HRP-linked antibody (Cell Signaling, Beverly, MA); HRP labeled goat anti-mouse IgG (KPL, Gaithersburg, MD, USA). Detection was performed using ECL (Pierce, Thermo Scientific), LiteAblot Extend Long Lasting Chemiluminescent Substrate (Euroclone) or LiteAblot Turbo Extra Sensitive Chemiluminescent Substrate (Euroclone) according to manufacturer's instructions.

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).