Ecl western blotting analysis system

For immunoblotting, extracellular vesicles were isolated from patients’ blood according to the protocol described above. After the ultracentrifugation step, the supernatants were removed, and the EV-containing pellets were resuspended in a proper volume of 1X RIPA buffer (150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS; 50 mM Tris-HCl, pH 8.0) supplemented with protease inhibitor cocktail (Roche, Penzberg, Germany). EV protein concentrations were quantified using a Bradford assay kit (ThermoFischer Scientific, Waltham, MA, USA). Twenty micrograms of EV protein lysates were separated to 4–10% SDS-PAGE using beta-mercaptoethanol as the reducing agent and transferred to nitrocellulose membranes (Amersham Biosciences, Amersham, UK). The membranes were then blocked in 5% nonfat dry milk in TBS-T (0.2% Tween-20) at RT and incubated overnight with the primary antibody against exosomal TSG101 (4A10, 1:500, Abcam, Cambridge, MA, USA). Immunoreactive bands were visualized with chemiluminescence using the ECL Western Blotting Analysis System according to the manufacturer’s instructions (Amersham Biosciences, Amersham, UK).

Cell lysates were prepared in a lysis buffer (Tris–HCl 20 mM pH 7.4 containing 1% Triton X-100, 0.1% SDS, 50 mM NaCl, 2.5 mM EDTA, 1 mM Na₄P₂O₇ 10H₂O, 20 mM NaF, 1 mM Na₃VO₄, 2 mM Pefabloc and Complete from Roche). Proteins were separated on a 7.5% SDS-polyacrylamide gel (Mini Protean III, BioRad, Richmond, Ca) and transferred for 2 hours at 4ºC to nitrocellulose membrane (Transblot Transfer Medium, BioRad, Richmond, CA) that was stained with Ponceau-S red as a control for protein loading. The membrane were incubated at 4ºC with rabbit polyclonal anti-rat VE-Cadherin (Abcam plc, Cambridge, UK) overnight in a 1:1000 dilution. Then, the membrane was incubated with goat anti-rabbit peroxidase-conjugated secondary antibody at a 1:5000 dilution (Cell Signaling, Beverly, MA) for 1 hour at room temperature. The bands were visualized by chemiluminescence (ECL western blotting analysis system; Amersham Biosciences).

Cells were lysed for 10 min at 99°C in 62.5 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol, 50 mM dithiothreitol, and 0.01% (w/v) bromophenol blue. Proteins were separated by SDS-PAGE and blotted onto PVDF-membranes (Roth, Karlsruhe, Germany). After blocking with 5% (w/v) non-fat dry milk, membranes were incubated at 4°C over night with the respective primary antibody (1:20,000 for β-actin, 1:1000 for all other antibodies). After washing, the membranes were incubated for 1 h at RT with the secondary antibody (anti-IgG-HRP 1:2000, Amersham-Biosciences, Freiburg, Germany), washed again, and developed using enhanced chemiluminescence staining (ECL western blotting analysis system, Amersham-Biosciences, Freiburg, Germany). We indicated that protein levels were quantified by densitometry using ImageJ software (ImageJ 1.40g, NIH, USA). The respective protein levels were normalized to β-actin levels.

Whole-cell extracts were made as previously described (Engedal et al, 2002 (link)), resolved by SDS–PAGE and transferred to a PVDF membrane (Bio-Rad). The blotted membrane was blocked in 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween (TBS–Tween) for 1 h followed by incubation with primary antibody in TBS–Tween containing 5% bovine serum albumin (BSA) for 14–16 h at 4°C. Antibodies used were against IRE1α (3294S), phospho-PERK (3179S), PERK (3192S) phospho-eIF2α (9721L), eIF2α (9722S), phospho-JNK (9251L), JNK (9252), ATF4 (11815S), cleaved caspase-3 (9661L), PCNA (13110S) (Cell Signaling), XBP-1 (sc-7160), CHOP (sc-7351), PSA (sc-7638), β-actin (sc-58670), GAPDH (sc-47724), β-tubulin (sc-9104) (Santa Cruz), α-tubulin (Sigma-Aldrich), AR (06-680) (Upstate), and phospho-IRE1α (PA1-16927) (Thermo Scientific). The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (Sigma-Aldrich) secondary antibodies in 5% nonfat dry milk dissolved in TBS–Tween for 1 h at room temperature. ECL Western blotting analysis system was utilized for detection of the immunoreactive bands according to the manufacturer's instructions (Amersham Pharmacia Biotech).

The protein lysates were prepared and Western blotting was conducted as previously described [35 (link)]. All antibodies (see Supplementary Table S2) were diluted in 5% milk/TBST, except for anti-pMLC2, which was diluted in 5% BSA/TBST. The signals were detected with the ECL Western blotting analysis system (Amersham Biosciences, Piscataway, NJ, USA).

Ovaries of adult females of each genotype were collected in 1× PBS, and then SDS-PAGE (five pairs of ovaries per lane) and Western blotting were performed as described earlier [18 (link)] with some modifications. An equal volume of 2× sample buffer with β-mercaptoethanol was added to the samples and mixed well by pipetting; the mixture was heated for 5 min and loaded onto an SDS-containing 5–15% gradient polyacrylamide gel. Proteins were separated at 250 mA for 2 h and next transferred onto a Hybond ECL membrane (Amersham Pharmacia Biotech, Amersham, UK, cat. # RPN2020D). For Western blotting, the membranes were incubated overnight at 4 °C with primary antibodies (mouse anti-NICD, DSHB, cat. # C17.9C6, at 1:40 dilution and mouse anti-alpha Tubulin, Novus Biologicals, Littleton, CO, USA, cat. # NB100-690, at 1:5000 dilution). After washing, the membranes were probed for 1 h with a secondary antibody (a goat anti-mouse IgG (H + L) antibody, horseradish peroxidase conjugate, Invitrogen, cat. # G21040, at 1:10,000 dilution) at room temperature. The signals were detected using the ECL Western Blotting Analysis System (Amersham Pharmacia Biotech, cat. # RPN2108) on an Amersham Imager 600 instrument (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

Protein was harvested using a RIPA buffer (Pierce, Rockford, IL, USA) and 1× protease and phosphatase inhibitors using standard techniques, and protein concentration was measured using the bicinchoninic acid (BCA) Protein Assay Kit (Fisher Scientific, Pittsburg, PA, USA). Twenty to thirty micrograms of total protein extract were separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Blotting was performed for procaspase-3 (31A893) (Abcam, Cambridge, UK), cleaved caspase-3 (Cell Signaling, #9662), and caspase-7 (Cell Signaling, #9492). β-actin (Abcam, Cambridge, UK) was used as a loading control. The secondary horseradish peroxidase (HRP) antibody was detected using ECL Western Blotting Analysis System (Amersham Biosciences, Piscataway, NJ, USA).