Fluorophorestar 3000

We analyzed the BALF proteome in patients with acute and chronic HP in five cases of each with 2‐DE. The proteins were evaluated as described previously.⁸, ¹³ The gels were scanned using a FluoroPhoreStar 3000 image analysis system (Anatech) and analyzed with Progenesis PG220 Software (Nonlinear Dynamics Ltd.). The proteins were analyzed automatically using the spot detection feature of the software, with automatic warping and matching. Spot volumes were corrected for background and then normalized. The normalized spot volume was calculated as a percentage of the volume of each spot to the volumes of all spots in a gel. Proteins were identified by liquid chromatography nano‐electronspray ionization tandem mass spectrometry (LC‐nESI‐MS/MS) in the Laboratory of Cytometry and Proteome Research at Tokyo Medical and Dental University. LC separation was performed by the nano‐UHPLC system (BrukerDaltonics). Mass analysis was performed on a maXis‐4G‐CPR (BrukerDaltonics) mass spectrometer equipped with a nano‐ESI source.

Protein or phosphoprotein gel staining and image acquisition were carried out as previously described [21 (link), 22 (link)]. Briefly, the gels were fixed three times in 200 ml immobilization solution [10% acetic acid and 50% methanol] for 30 min and washed five times with 200 ml of water for 15 min. Under the dark, the gels were stained with Pro-Q Diamond phosphoprotein gel stain (Life Technologies, Carlsbad, CA) for 120 min at room temperature with gentle agitation and then washed three times with destaining solution [50 mM sodium acetate, pH 4.0 and 20% (v/v) acetonitrile] for 30 min. Image acquisition was performed on Fluorophorestar 3000 image capture system (Anatech, Tokyo, Japan) with a 520 nm excitation and 575 nm emission filter for Pro-Q Diamond detection.
Next, gels were washed with washing solution [10% methanol and 7% acetic acid] for 30 min. The gels were incubated in SYPRO Ruby stain (Life Technologies) for 90 min in the dark. The gels were washed with destaining solution [10% methanol and 7% acetic acid] for 30 min and rinsed with MilliQ H₂O. Image acquisition was carried out using a Fluorophorestar 3000 image capture system with a 470 nm excitation and 618 nm emission filter for SYPRO Ruby detection.

FLAG-tagged RBM20 proteins expressed in HEK293T cells or immuno-precipitated and subsequently treated with CiAP and λPP were separated by neutral polyacrylamide gel electrophoresis (NuPAGE, Invitrogen). The NuPAGE gel was stained with Pro-Q Diamond (Molecular Probes) according to the manufacturer’s instruction, and fluorescence images were acquired with FluoroPhore Star3000 (Anatech). The gel was then stained with Gel-Negative Staining Kit (Nacalai). For Phos-tag SDS-PAGE, a 15% polyacrylamide gel without or with 25 µM Phos-tag (Wako) and 50 µM MnCl₂ was prepared and layered with a standard 4.5% stacking gel according to the manufacturer’s instruction. Protein samples were run with standard SDS running buffer (Nacalai). Vertical SDS-agarose gel electrophoresis of cardiac proteins from mice were performed essentially as described previously^{37 (link)}. The proteins were detected by staining with CBB (Bio Craft). The images of the stained gels were captured with a scanner GT-X700 (Epson) and processed by using Photoshop CC (Adobe).