Amersham imager 600rgb

Proteins were selected for validation by western blot analysis based on significance as well as function. Proteins were separated on an AnyKD SDS-PAGE gel (BioRad) and transferred to a PVDF membrane using the Trans Turboblot system (BioRad). Membranes were blocked in 5% non-fat milk in TBS for 1 hour at room temperature. Primary antibodies specific for phospho-Serine47-Histone H4 (rabbit polyclonal, 1:500), Histone H4 (mouse monoclonal, 1:1000), PCTAIRE-2 and PCTAIRE-3 (rabbit polyclonal, 1:1000), actin (mouse monoclonal, 1:7000) and GAPDH (rabbit monoclonal, 1:5000) were diluted in 5% BSA-TBS, containing 0.05% sodium azide and incubated overnight at 4°C. Membranes were washed and then incubated with appropriate corresponding secondary antibodies, donkey anti-rabbit HRP-conjugated or goat anti-mouse HRP-conjugated for 1.5 hours at room temperature. After further washes, all blots were developed with Pico Chemiluminescence reagents, with the exception of pS47-Histone H4 which was developed using Femto Chemiluminescence reagents, using an Amersham Imager 600RGB (GE Healthcare).

Expression of prolyl-4-hydroxylase (PDH4) was evaluated by immunofluorescence assay (IFA) and Western blot (WB). For IFA, cells were seeded into fixed and permeated chamber-slides. Subsequently, primary antibody PDH4 (ab108980, Abcam) was incubated. Afterwards, secondary antibody (ab175471 Alexa Fluor® 568, Abcam) was incubated. Finally, images were captured with an Ism 5 beta Carl Zeiss microscope.
Total protein was obtained from the culture of sinoviocytes. Analysis of the protein content was performed by WB according to Serratos et al. [15 (link)] Normalization was performed with Beta-actin antibody from Sigma (A3854). Blots were revealed using Immobilon Western Chemiluminescent HRP Substrate (Millipore Corporation, USA). The blots were scanned with an Amersham Imager 600 RGB (GE) and densitometry was analyzed using ImageQuant TL 8.1 software.

Gels after SDS–PAGE were fixed in solution containing 50% (v/v) ethanol and 10% (v/v) acetic acid. On the following day, the gels were washed three times with ddH₂O for 10 min and stained with Pro-Q Diamond phosphoprotein stain (Invitrogen) for 90 min. The gels were washed three times with ddH₂O for 10 min, destained twice for 30 min in 50 mm sodium acetate, pH 4.0, containing 20% (v/v) acetonitrile and finally washed with ddH₂O. Fluorescent bands on the gel were visualized with an Amersham Imager 600RGB (GE Healthcare).

NK-92 cells were treated with UMCD6 or an IgG control antibody at 10 μg/ml and cell lysates were collected after 72 hours. To measure changes in PI3K and mTOR expression, HRP-conjugated antibodies to PI3K (Cell Signaling, Cat#428T), and mTOR (Cell Signaling, Cat#2972) were used at 1:1000 in 5% milk. β-actin (Cell Signaling, Cat#13E5) was used control for loading. Bands were imaged on an Amersham Imager 600RGB (GE Healthcare).

After incubation of NR8383 cells with different silica particles (100 µg mL⁻¹) for 16 h, the supernatants were centrifuged at 300 g for 10 min and stored at -20 °C until the microarray analysis (Profiler Array Rat XL Cytokine Array Kit, Bio-Techne GmbH, Wiesbaden-Nordenstadt, Germany). The assay detected 79 different cytokines, growth factors and other mediators and enabled a semiquantitative analysis. The membrane-based sandwich immunoarray consists of a nitrocellulose membrane on which the captured antibodies are spotted as duplicated dots. Target proteins present in the sample bind to the capture antibodies and are detected with biotinylated detection antibodies and then visualized with chemiluminescent detection reagents. For analysis, the manufacturer's instructions were observed and chemiluminescence signals were detected and quantified by a microarray imager and the ImageQuantTL software (Amersham Imager 600 RGB, GE Healthcare Bio-Science, Uppsala, Sweden). For subsequent detailed analysis using Sandwich-ELISA Kits (R&D Systems Quantikine, Bio-Techne GmbH, Wiesbaden, Germany), 27 factors were selected based on the proteomic repertoire of NR8383 cells, as reported by Duhamel et al.^{58 (link)}.

Whole protein samples were prepared from culture samples taken at specific time points after induction by addition to ice-cold 10% (vol/vol) trichloroacetic acid. Pelleted protein samples were then washed with cold acetone before resuspension in nonreducing Laemmli buffer. Electrophoresis on Novex 16% Tricine gels (Thermo Fisher Scientific, Waltham, MA) was performed according to manufacturer recommendations. Proteins were transferred to a 0.2-μm polyvinylidene difluoride membrane by the iBlot2 system with a pre-programmed protocol (20 V for 1 min, 23 V for 4 min, 25 V for 1 to 2 min). After blocking with 4% (wt/vol) milk–Tris-buffered saline; His-tagged and c-myc tagged proteins were detected separately. Antibodies and substrate were acquired from Thermo Fisher Scientific (Waltham, MA). The mouse α-His primary antibody was used at a 1:2,000 dilution, and the secondary goat-anti-mouse Alexa Fluor 680 was used at a 1:20,000 dilution. α-c-myc (9E10) mouse monoclonal was used at a dilution of 1:1,000 overnight, followed by secondary antibody (goat-anti-mouse horseradish peroxidase) at 1:1,000, with SuperSignal West Femto Maximum Sensitivity Chemiluminescent Substrate (Thermo Fisher) for detection. Blots were scanned on Amersham Imager 600 RGB (GE Healthcare, Chicago, LA) and Bio-Rad-ChemiDoc machines, respectively, and analyzed using LI-COR ImageStudioLite version 4.0.21 software.