Nupage bis tris gradient gel

Basal and activated platelets (8 × 10⁸platelets/mL, 500 μL per immunoprecipitation; activations with CRP-XL 1 μg/mL, 90sec) were lysed with NP40-based lysis buffer, as indicated in Supplementary Material. Activations were under non-aggregating conditions in the presence of integrilin (9 μM). Phosphotyrosine (p-Tyr) immunoprecipitations were done with 5 μg of agarose-conjugated 4G10 monoclonal anti-phosphotyrosine antibody (EMD Millipore Corporation, Billerica, MA, USA) per sample, as previously shown [13 (link)]. Further details on the protocol can be found in Supplementary Material.
Immunoprecipitations from twelve obese patients and ten lean matched-controls were loaded independently, and proteins resolved on 4–12% NuPAGE Bis-Tris gradient gels (Invitrogen, Carlsbad, CA, USA). Following the electrophoresis, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (GE Healthcare) and incubated with primary antibodies (PLCγ2 and G6F) as describe above.

QFWB was carried out as previously described (21 (link), 22 (link)). Briefly, 15 μg of CCG protein were separated by SDS-polyacrylamide gel electrophoresis on 4–12% precast NuPage BisTris gradient gels (Invitrogen, Paisley, UK) and then transferred to PVDF membrane using an iBLOT fast transfer device (Invitrogen). The membranes were then blocked using Odyssey blocking buffer (LICOR Biosciences, Cambridge, MA) and incubated with primary antibodies according to manufacturers' instructions (see Table II). Odyssey secondary antibodies were added according to manufacturers' instructions (goat anti-rabbit IRDye 680 and goat anti-mouse IRDye 800). Blots were imaged using an Odyssey Infrared Imaging System (LI-COR Biosciences). Scan resolution of the instrument ranges from 21 to 339 μm, and blots were imaged at 169 μm. Quantification was performed on single channels with the analysis software provided. Total protein stain gels, loaded in parallel with those used for membrane transfer, were used to ensure equivocal sample loading and were analyzed using the Odyssey Infrared Imaging System as previously described in (21 (link), 22 (link)).

6xHis-Rad6, 6xHis-Ubc4, Ubi-ProtA-6xHis, and Ubi^K48R-ProtA-6xHis were expressed in E.coli BL21(DE3) pRIL and purified over a pre-packed HisTrap FastFlow column (GE Healthcare). FLAG-Ufd4, FLAG-Ubr1, FLAG-Ufd2, and FLAG-Tom1 were overexpressed in yeast from a GPD promoter and purified as described (Hwang & Varshavsky, 2008 (link); Hwang et al, 2009 (link)). Purified yeast Uba1 and ubiquitin were purchased from BostonBiochem (#E-300 and #U-100SC, respectively). Final protein concentrations were 100 nM (Uba1), 80 μM (ubiquitin), 1 μM (Rad6), 1 μM (Ubc4), 200 nM (Ubr1), 200 nM (Ufd4), 200 nM (Ufd2), 200 nM (Tom1), 125 nM (Ubi-ProtA), 125 nM (Ubi^K48R-ProtA), in 20 μl reactions containing 4 mM ATP (#1191, Merck), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 50 mM Hepes (pH 7.5). All reactions contained Uba1, ubiquitin, Rad6, and Ubc4. Reactions were pipetted on ice, incubated at 30°C for 30 min, quenched by addition of 20 μl 5× SDS sample buffer (50% [vol/vol] glycerol, 10% [wt/vol] SDS, 250 mM Tris–HCl, pH 6.8, 62.5 mM EDTA, and 5% [vol/vol] β-mercaptoethanol) and incubation at 95°C for 5 min, and analyzed using 4–12% NuPAGE Bis-Tris gradient gels (Invitrogen) followed by immunoblotting with rabbit peroxidase–anti-peroxidase (PAP) antibodies (Z0113; Dako) and imaging on a LAS-4000 system (Fuji).