Gradient sds page gel

Eluted proteins were separated on 4–12% gradient SDS-PAGE gels (Invitrogen) and stained with Colloidal Coomassie Blue. Each gel lane was cut into 23 equal gel slices and proteins therein were in-gel digested with trypsin as described57 (link). Tryptic peptides from each gel slice were analysed as described58 (link) by nanoflow HPLC (Agilent 1100, Agilent Technologies) coupled to nanoelectrospray LTQ-Orbitrap XL mass spectrometer (Thermo Fischer Scientific). Raw MS data files were analysed by MaxQuant (version 1.3.0.5)22 (link) and Andromeda59 (link) using the UniProt rat protein database (version 05.13) and with selected “label-free protein quantification” (LFQ). Results from MaxQuant were further processed using Perseus (version 1.3.0.4, www.perseus-framework.org). Contaminant and reverse entries were filtered and all LFQ intensities were logarithmised. Missing values were imputed with random numbers using normal distribution (width = 0.3, down shift = 1.8) to simulate low abundance intensity values21 (link). A modified t test (250 permutations, FDR = 0.01, S₀ = 2)60 (link)61 (link) was applied to identify proteins significantly enriched within each IP experiment using four biological replicates and compared to identical control IP.

Exosomes isolated from serum were suspended in 50 μl M-PER reagent (Thermo Scientific) with HALT protease inhibitor cocktail (Thermo Scientific). After determining the protein concentration using Bradford analysis, the samples were fractionated on a 4–12% gradient SDS-PAGE gels (Invitrogen, Carlsbad, CA). Subsequently, the gels were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was incubated with anti-CD63 (H5C6) (Novus Biologicals, Centennial, CO) or anti-CD9 (D3H4P) antibodies (Cell Signaling Technology, Danvers, MA) overnight at 4 °C, followed by incubation with peroxidase conjugated anti-mouse/-rabbit antibody. The proteins were visualized using an Immobilon Forte Western HRP blotting substrate (Millipore, Burlington, MA).

A bullet blender (Next Advance) was used to homogenize cell lysates. Protein concentrations were normalized by DC™-protein assay (Bio-Rad), and equal amounts of protein were loaded into 4–12% gradient SDS PAGE gels (Invitrogen). After electrophoresis and transfer onto a PVDF membrane, immunoreactivity was detected by ECL (Thermo Scientific) and imaged using an iBright (Thermo Fisher Scientific). ImageJ was used to quantify band intensity [25 (link)]. For Endoglycosidase H assay, lysates were separated into three reactions containing: negative control (NT), EndoH (E) (NEB P0702L), or PNGaseF (P) (NEB P0704L) [25 (link)]. After a 3-h incubation at 37 °C, samples were loaded on SDS PAGE gels as indicated above.

HEK293 cells were plated in 6-well plates and then incubated with Flag-tagged SF-1 and HA-tagged ubiquitin. One day after transfection, cells were treated with MG132 at 25 μM for 6 hours in the presence or absence of serum. Cells were harvested in RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.5% sodium deoxycholate, 1× protease inhibitor cocktail and 1× phosphatease inhibitor cocktail (Roche)) and centrifuged at maximum speed for 20 min at 4 °C. Equal amounts of total protein were incubated with 2 μl anti-SF-1 specific antiserum12 (link) overnight at 4 °C and then added to a protein G-Sepharose bead slurry for 2 hours at 4 °C. The final immune complexes were analyzed by Western blot.
For Western blots, proteins were resolved on 4–12% gradient SDS-PAGE gels (Invitrogen) and transferred to Nitrocellulose membranes (Bio-Rad). Target proteins were detected using anti-SF-112 (link), anti-HA (F-7) or anti-GAPDH (Santa Cruz Biotechnology), anti-Flag, anti-GFP or anti-Actin (Sigma), or anti-phospho-AKT (S473, Cell Signaling) antibodies. The immune complexes were visualized with an Enhanced Chemiluminescence (ECL) detection kit (Pierce) or an Odyssey IR imaging system (LI-COR Biosciences).

Superdex 200 and Superdex 75 prepacked columns were obtained from GE Healthcare Life Sciences, Chicago, USA. The 4–12% gradient SDS-PAGE gels were purchased from Invitrogen and Bradford’s reagent was obtained from Pierce. All other chemicals were of analytical grade.