Bis 1 3 dibutylbarbituric acid trimethine oxonol

Fura-2 acetoxymethyl ester (Fura-2/AM) was from Calbiochem (La Jolla, CA, USA). Bis-(1,3-dibutylbarbituric acid) trimethine oxonol, DiBAC₄-(3), was obtained from molecular probes (Eugene, OR, USA). All reagents, including materials for video imaging experiments, were purchased from Sigma Chemical Co. (St. Louis-MO, USA).
Primary antibodies against: Calnexin (H-70), GAPDH (FL-335), CaMKII (M-176), pCaMKII (Thr286), p70 S6 (C-18), pp70 S6 (C-18), ERK1 (K-23), ERK2 (C-14), pERK1/2 (E-4), OPN (K-20), peroxidase-conjugated secondary antibodies for western blot analysis and FITC or Rhodamine-conjugated antibodies for immunofluorescence study were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). AlexaFluor^®488-Phalloidin was obtained from Life Technologies (Carlsbad-CA, USA).

The easy select expression kit for expression of recombinant proteins using pPICZα in P. pastoris and zeocin were obtained from Invitrogen (Carlsbad, CA, USA). Bis-(1,3-dibutylbarbituric acid) trimethine oxonol was acquired from Molecular Probes^® (Part of Life technologies; Carlsbad, CA, USA). Yeast nitrogen base (YNB), dithiothreitol, S-(5′-adenosyl)-l-methionine, epinephrine (bitartrate salt), deoxyribonuclease (DNase), protease inhibitor cocktail, dl-metanephrine hydrochloride, glass beads (500 µm) and propidium iodide were purchased from Sigma-Aldrich (St. Louis, MO, USA). All chemicals used were of analytical grade, commercially available, and used without further purification.
E. coli TOP10F’ was used for DNA manipulations. E. coli transformants were selected on low-salt Luria–Bertani plates with 25 µg/mL Zeocin. P. pastoris X-33 and KM71H was used for fusion gene expression. YPD and YPDS media [40 ] were used for routine manipulation of Pichia cells. P. pastoris transformants were selected on YPDS plates with 200 µg/mL Zeocin. Small-scale fermentations were carried out in BMGH and BMMH media [40 ]. P. pastoris bioreactor cultures were carried out in modified basal salts medium (BSM) [27 (link)] with 200 µg/mL zeocin and supplemented with trace metal solution (SMT) [27 (link)].