Imagequant rt ecl

Isolated melanosomal fractions were introduced to 1% Triton-X-100 buffer containing inhibitors (PMSF and protease inhibitor cocktail, Sigma-Aldrich) and disrupted with sonication. Twenty μg of protein was loaded on each well of SDS-PAGE gel (4–20% Precise Tris-Glycine Gels, Thermo Fisher Scientific Inc. Waltham, MA, USA). Proteins were transferred onto PVDF membrane (Bio-Rad) with semi-dry electroblotting (Trans-Blot^® SD Semi-Dry Transfer Cell, Bio-Rad). Primary antibody incubations were conducted at +4°C overnight (mouse-anti HSP60 1:1000, #ADI-SPA-806-F, Enzo Life Sciences Inc., Farmingdale, New York, USA; Anti-Na⁺/K⁺ATPase α1, 1:1000, 05–369, Merck Millipore, Merck KGaA, Darmstadt, Germany; Atp6v0a1 antibody, 1:500, GTX44653, GeneTex Inc., Irvine, CA, USA, Rab27a antibody 1:500, 0023, SICGEN–Research and Development in Biotechnology Ltd, Cantanhede, Portugal). Secondary antibody incubations (goat anti-mouse, sc-2005 and goat anti-rabbit sc-2030, 1:10 000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) took place at room temperature for 45 min. Protein-antibody-complexes were detected with chemiluminescence reaction (Image Quant RT ECL, GE Healthcare, Little Chalfont, UK) using Amersham^™ ECL^™ Prime Western Blotting Detection Reagent (GE Healthcare).

Arrestin-mCherry fusion proteins were expressed, harvested and lysed as described^{14 (link)}. The cell lysate from a 50-mL cell-culture fraction was cleared by centrifugation (Centrifuge 5424 R; Eppendorf) at 21,100 × g for 20 min at 4 °C. The lysate was distributed in 100-μL portions to eleven 1.5-mL tubes (Sarstedt), which were put into a heating block that was equilibrated at 30 °C. The temperature was ramped up to 80 °C in 5 °C-steps manually at 2.5 min intervals. Samples were removed successively every 2.5 min and chilled on ice. Precipitant was removed by centrifugation for 1 h. 12 μL supernatant of each sample was mixed with 3 μL 5x SDS-loading dye. Full-length arrestin-mCherry construct was separated from degraded protein by SDS-PAGE for 1 h at 200 V and 80 mA in MOPS buffer using an 8–12% Bis-Tris gradient gel (Novex NuPAGE; Life Technologies) in supplied chamber (Novex NuPAGE SDS-PAGE gel system; Life Technologies). Fluorescence-emission of mCherry or mCherry-protein fusions was detected by exciting the protein at 312 or 365 nm using a 605 nm-filter (ImageQuant RT ECL; GE Healthcare). Fluorescence intensities were quantified by ImageJ (NIH) and plotted in Prism. Boltzmann sigmoidal fitting allowed to determine T_M and R² values and standard errors.

Whole cell extracts were prepared using lysate buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP40, and 0.1% SDS) with a Protease and Phosphatase Inhibitor Cocktail (Nacalai). Protein concentrations were measured using the BCA assay (Takara Bio, Shiga, Japan). Samples were diluted in 5 × SDS buffer (0.225 M Tris–HCl, 50% glycerol, 5% SDS, 0.05% BPB, and 0.25 M DTT) and treated at 95°C for 5 min. Proteins (20 μg/lane) were separated by 8 ~ 10% SDS‐PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. Non‐specific binding sites on PVDF membranes were blocked with 5% Skim milk (Yukijirushi) for 1 h. Membranes were incubated with antibodies against HER3, MET, phospho‐HER3, phospho‐MET, phospho‐ERK, ERK, FOXM1, and GAPDH (CST) for 16 h, and then with horseradish peroxidase‐labeled goat anti‐rabbit or mouse IgG (H+L) pAb (1:400, #115‐035‐144 or #115‐035‐062, Jackson ImmunoResearch). Immunoreactive proteins were visualized by an ECL system (ImageQuant RT ECL, GE Healthcare) using Chemi‐Lumi One Super (Nacalai) in Kindai University, and by exposing X‐ray film (Super RX, Fujifilm, Tokyo, Japan) using a detection solution (Hi‐RENDOL, Hi‐RENFIX, Fujifilm) or by an ECL system (ChemiDoc, BioRad, Hercules) in Tohoku University.

EMSA assays were performed using cell lysates from PAO1, pqs mutants, and Rosetta strains induced for His-SUMO or His-SUMO-PqsR production as described (Cugini et al., 2007 (link)). For each sample, 10 µg of cell-lysate was incubated with 0.15 fmol of biotinylated 248-bp pqsA promoter DNA (pqsA’), which was generated by PCR using biotinylated forward primer (TTCTTGCTTGGTTGCCG) and reverse primer (GACAGAACGTTCCCTCTT). Unlabeled pqsA’ probe was generated using the same primers as the biotinylated-pqsA’ without the biotin modification. The reaction mixture contained 10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 50 mM NaCl, 1 mM DTT, 1 µg/µl Poly(dI-dC) and was incubated at room temperature (24 °C) for 20 min. Samples were separated on a 5% polyacrylamide gel in 0.5×Tris Borate EDTA (TBE) run at 100 V for 50 min and transferred onto a positively-charged nylon membrane at 40 V for 1 h. Biotinylated DNA on the nylon membrane was probed by streptavidin-HRP conjugate, detected by a chemiluminescent substrate (Pierce LightShift Chemiluminescent EMSA kit; Thermo Fisher Scientific, Waltham, MA, USA), and visualized by exposing to either an X-ray film or GE ImageQuant RT-ECL.

Four adult flies of the corresponding feeding condition were lysed in 50 μl of EBR buffer [2 mM of Cl₂Ca, 129 mM of NaCl, 4 mM of KCl, and 35 mM of Tris (pH 6.85)] supplemented with phosphatase/protease inhibitor cocktail (Sigma #P2714), NaF (50 mM), and sodium orthovanadate (1 mM). A 12 mg protein/lane and a 24 mg protein/lane were used for pERK and pAKT immunoblots, respectively. Tissue samples were run on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene difluoride (PVDF) (Thermo Scientific). Primary antibodies used were as follows: anti-pAKT^Ser505 1:1,000 (Cell Signaling #4054), anti-AKT 1:1,000 (Cell Signaling #9272), anti-pERK1/2 1:1,000 (Sigma #M8159), and anti-ERK1/2 1:15,000 (Sigma #M5670). Horseradish peroxidase (HRP)-conjugated secondary antibody goat anti-mouse 1:1,000 (Sigma #A5278), goat anti-rabbit 1:5,000 (Sigma #A0545), and Super Signal West Pico (Thermo Scientific) were used for signal detection in an ImageQuant RT ECL (GE Healthcare Life Sciences) device. Activation was quantified by ImageJ 1.47v as phospho-protein normalized to non-phospho-protein (pERK/ERK and pAKT/AKT).