Immun blot

Total cellular protein was obtained from about 107 cells from each sample, using RIPA buffer. Lysates were incubated on ice for 15 minutes and then centrifuged for 10 minutes at 14,000 rpm. Protein extracts were then separated by SDS-PAGE electrophoresis on acrylamide 5% stacking and 7.5% separating gels, using the Mini-Protean electrophoresis cell (BioRad), according to standard procedures. Molecular weight values were estimated using prestained protein markers (Full Range Rainbow Marker, GE Healthcare). Proteins were transferred from the gel to PVDF membrane (Immun-blot, Biorad), according to the manufacturer's instructions. Membranes were then incubated in blocking solution (5% w/v BSA in TBS-T, i.e., Tris-buffered saline/0.1% Tween 20) for 1 hour at room temperature and the primary antibody was added at a dilution 1/1000 (PARP rabbit mAb, Cat. number 9542, Cell Signal, or β-actin rabbit polyclonal Ab, Cat. number 4967, Cell Signal, when membranes were reprobed for loading control). After overnight incubation at 4°C, the membrane was washed 3x in TBS-T and incubated with secondary antibody at a dilution 1/4000 in blocking buffer for 1 h at room temperature (anti-rabbit IgG, HRP conjugated, Cat. number 7074, Cell Signal). After 3x washes in TBS-T, signal was detected with ECL Blotting reagent (GE Healthcare) and X-OMAT LS-1 film (Kodak).

Triplicate MEC1 protein samples (15 μg) from each condition were separated by 10% SDS-PAGE, and transferred to a PVDF membrane (Immun-Blot™, Bio-Rad Laboratories, Hercules, CA, USA). After blocking with 5% skim milk in TBST (25 mM Tris/HCl, 137 mM NaCl, 2.7 mM KCl and 0.1% (v/v) Tween-20, pH 7.6), the membranes were incubated in TBST (4°C, 16 h) with rabbit monoclonal antibodies against BRCA1, NCL, NFKB2 p100/p52 or MYC (Cell Signalling Technology, Danvers, USA) or mouse monoclonal antibody against CCND1 (Cell Signalling Technology) and actin (Abcam, Cambridge, USA), followed by incubation (1 h, room temperature) with horse radish peroxidase (HRP)-conjugated secondary antibodies: goat-anti-mouse-HRP (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or donkey-anti-rabbit-HRP (Abcam, Cambridge, UK). Proteins were visualized using Rapid Step ECL Reagent (Merck, Kilsyth, Victoria, Australia) and ECL chemiluminescence film (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Films were scanned on a Molecular Imager GS-800™ densitometer (Bio-Rad, Hercules, CA, USA). Bands were quantified using ImageQuantTL density analysis software (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and two-tailed homoscedastic student t-tests were performed on log₂-transformed, actin-normalized ratios to the control samples.

Whole cell extracts were prepared using radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) with protease and phosphatase inhibitors (cOmplete - protease inhibitor cocktail tablets, and PhosSTOP - phosphatase inhibitor cocktail tablets; Roche), and total protein concentrations were determined by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Rockford, IL). Cell lysates containing equal amounts of total protein (∼5–10 µg) were heated at 100°C for 5 minutes in laemmli buffer (Sigma-Aldrich), electrophoretically separated on a 10% SDS-polyacrylamide gels (Criterion Precast Gel; Bio-Rad, Hercules, CA), and transferred onto polyvinylidene difluoride (PVDF) membranes (Immun-Blot; Bio-Rad). Membranes were incubated with primary antibodies for phospho-GSK3β-Ser9 (p-GSK3β-S9; Cell Signaling Technology, Danvers, MA; 5558), GSK3β (t-GSK3β; Cell Signaling Technology; 9832) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Abcam; ab8245). Appropriate horseradish peroxidase-conjugated secondary antibodies (SouthernBiotech, Birmingham, AL) were used. Membranes were detected using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific), and visualized using a Kodak Image Station 440CF.

Exosomal protein was extracted in RIPA Buffer in the presence of protease inhibitors, separated by a 12% SDS-PAGE and transferred to a PVDF membrane (Immun-Blot, Bio Rad, Hercules, CA). Membranes were blocked for 1 h in 5% nonfat milk in PBS, followed by overnight incubation with antibodies against Alix and TSG101 as well as Calnexin as negative control (Acris antibodies, Herford, Germany). Blots were washed and incubated for 1 h 30 min at room temperature with a DyLight 800-conjugated secondary antibody. Immunoreactive bands were visualized with an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).