Quantity one imaging system

Cells were plated on six-well plates, grown to 80% confluence, rinsed with serum-free DMEM or DMEM/F12 (v/v) and incubated overnight in serum-free medium. For PTX treatment, the cells were pretreated with 100 ng/mL PTX overnight prior to the MAPK assay. Cells were preincubated with various inhibitors for indicated time before activation with the indicated ligands. Ligand incubation was ended by washing the cells with 2 ml of ice-cold PBS followed by the addition of RIPA lysis buffer at 4°C on a rocker for 30 min. The lysates were centrifuged at 4°C at 12,000 rpm for 15 min. The supernatants underwent electrophoresis on a 10% SDS polyacrylamide gel, which was transferred to a PVDF membrane and immunoblotted using monoclonal anti-phospho-ERK1/2 (Thr202/Tyr204) (1∶1000) or anti-phospho-IGF-1Rβ antibody (1∶500). Blots were probed with horseradish peroxidase-labeled secondary antibodies, and chemiluminescence was detected using HRP-substrate (Cell Signaling). The blots were stripped and reprobed using an anti-total ERK1/2 (1∶2000) or anti-α-tubulin monoclonal antibody as a control for protein loading. The levels of ERK 1/2 phosphorylation was normalized to total ERK1/2, and all the immunoblots were visualized and quantified by Bio-Rad Quantity One Imaging system (Bio-Rad Laboratories).

To detect protein expression levels, breast cancer cells were lysed in ice-cold RIPA lysis buffer (Biospes). Homogenates were then centrifuged at 12,000 × g for 20 min at 4°C. Protein content in the clear supernatant was quantified using a bicinchoninic acid kit (Beyotime), and samples were then reduced and stored at −80°C until use. Samples (20–50 μg protein/lane) were separated by SDS-PAGE. Proteins were transferred from the gel onto a PVDF membrane. After protein transfer, PVDF membranes were blocked with a solution containing Tris-buffered saline, 0.1% Tween 20 (TBST) and 5% fat-free milk for 2 h at room temperature. Membranes were then incubated with primary antibodies (See Table S1) overnight at 4°C. Afterwards, blots were treated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG (1:10,000) in TBST containing 1% (w/v) BSA for 60 min at room temperature while shaking, and immune complexes were detected using an ECL plus detection kit (Thermo). Finally, PVDF membranes were scanned using the Quantity One Imaging system (Bio-Rad). The relative expression of target protein was normalized to that of GAPDH or β-actin. Every experiment was repeated three times.

For determination of gelatinase activity of MMP-2 and MMP-9 in blood serum “Ready-Gelatin-Gels” (Bio-Rad, USA) were used according to the instructions of the manufacturer and as published earlier [22 (link)]. Two microliters from individual serum samples were electrophoresed under non-reducing conditions using Criterion™ Precast Gel System (Bio-Rad, USA). After electrophoresis, each gel was incubated at room temperature in 2% Triton X-100 for 2 × 30 min in order to remove the traces of sodium dodecyl sulphate, and then incubated overnight at 37 °C in buffer (150-mM NaCl, 50-mM Tris-HCl, pH 7.6, containing 5-mM CaCl₂ and 0.02% NaN₃). Afterwards a staining with 0.5% Coomassie blue G-250 (Sigma, USA) was performed for each gel. The proteolytic activity of each gelatinase (A and B) was identified as a clear band on a blue background according to the correspondent molecular weight of each gelatinase (A and B that corresponds to the metallproteinase-2 and -9, respectively). Gels were dried between cellophane sheets with a GelAir™ drying system (Bio-Rad, USA) and then scanned with a yellow filter using Adobe Photoshop (Adobe System, USA) in grey-scale mode. Densitometric analysis of zymographic lysis zones at 66 and 86 kDa for gelatinases A and B, respectively, was performed using Quantity One imaging system (Bio-Rad, USA).

For Western blot analysis, cells were scraped off, centrifuged, resuspended in lysis buffer (Urea 7 M, Thiourea 2 M, Chaps 4%, Tris 40 mM, PMSF, DTT), and sonicated for 1 min. Subsequently, protein concentrations were determined with the Bradford method using a commercial kit from BioRad. Equal amounts of protein were resolved on 10%-SDS polyacrylamide gels and blotted onto nitrocellulose membranes. Unspecific binding was blocked with 5% milk in 0.6% Tween-TBS (TTBS) for 1 h. Primary antibody was added in TTBS/5% dry milk and incubated overnight at 4 °C. For protein detection, blots were incubated with the secondary antibody in TTBS/5% dry milk, followed by ECL-detection of specific proteins using the Quantity One imaging system (Bio-Rad). Equal loading of protein was determined by visual detection by incubating the membranes with Ponceau. The sample treatment in the actin Western blot was performed to differentiate the F- and G-actin forms. The samples were homogenised in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 2 mM MgCl2, pH 6.9, containing 0.1% Triton X-100, 0.5 mM PMSF, and 0.1 mM DTT). The homogenate was centrifuged at 48,000 g during 5 min at 4 °C. The insoluble fraction (cytoskeleton associated) was used for Western blot analysis^{13 (link)}.

Western blot (WB) analysis was performed to determine protein levels of NF-κB, Toll-like receptor 4 (TLR4), β-actin, and GAPDH in the rat liver tissues. In brief, total proteins were isolated from the liver tissues of the experimental rats. After 80 μg of total proteins were separated on sodium dodecyl sulfate- (SDS) polyacrylamide gel electrophoresis (PAGE) gels, the gels were transferred onto polyvinylidene fluoride (PVDF) members, which were then blocked and incubated at 4°C overnight with primary antibodies including rabbit anit-mouse NF-κB polyclonal antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA), mouse anti-rat TLR4 polyclonal antibody from Abcam (Cambridge, MA, USA), rabbit anit-mouse polyclonal β-actin antibody from ZSGB-Bio (Beijing, China), and GAPDH antibody from Abcam (Cambridge, MA, USA). The specific bands were visualized and analyzed with Quantity One imaging system from Bio-Rad (Hercules, CA, USA). The intensity of NF-κB and TLR4 bands was normalized to β-actin or GAPDH.

Western blot was carried out as previously described [31 (link)]. Cells were lysed in RIPA buffer (with a cocktail of phosphatase and protease inhibitors) and solubilized in Laemmli buffer.
Amounts of 20 µg protein from cell lysates, determined by BCA Protein Assay Kit (Cayman Chemicals, Ann Arbor, MI, USA), were separated by SDS–PAGE, blotted to nitrocellulose membrane, and probed with one of the following primary antibodies (dilution 1:400): anti-ERO1α (cat# 12007-1-AP, ProteinTech Group, Chicago, IL, USA), anti-GRP78/BiP (cat# ab21685, RRID: AB_2119834, Abcam, Cambridge, UK), anti-CHOP/GADD 153 (cat# ab11419, Abcam), anti-XBP1 (cat# ab37152, Abcam). Membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Bethyl Laboratories, Montgomery, TX, USA; dilution 1:1000), developed by an ECL kit, acquired by ChemiDoc XRS (Bio-Rad Laboratories, Hercules, CA, USA), and digitized by Quantity One Imaging System (Bio-Rad). Equal loading was confirmed with anti-β-actin antibody (cat# A300-491A, Bethyl Laboratories, Montgomery, TX, USA).

Cells were treated with trypsin, removed from flasks and then collected by centrifugation. Cells were then lysed using RIPA lysis extraction buffer supplemented with Halt protease inhibitor cocktail and EDTA (Pierce-Thermo Scientific). Lysed samples were separated on a 4–12% NuPAGE Bis–Tris gradient gels and protein transferred on to nitrocellulose membranes by transverse electrophoresis. Nitrocellulose membranes were blocked in 3% (w/v) non-fat dried skimmed milk powder dissolved in PBS for 1 h at room temperature, and probed with primary antibodies including anti-IP₃R1 (1:500 dilution), anti-IP₃R2 (1:1000 dilution), anti-IP₃R3 (1:500 dilution) and anti-β-actin (1:1000 dilution) at 4°C overnight. After overnight incubation, nitrocellulose membranes were incubated with appropriate species-specific HRP-conjugated secondary antibodies (1:400 dilution) for 1 h at room temperature. Bands were visualized using enhanced chemiluminescence (ECL) Western blotting substrate and a Bio-Rad Quantity One imaging system. Band intensities were quantified and analysed using ImageJ software (NIH). Blotting for β-actin was used as a loading control for siRNA knockdown experiments.