Western Blotting of Protein Samples

Protein samples in 1× LDS sample buffer containing 4% BME were separated by size on 4 to 12% Novex NuPage Bis-Tris SPS-PAGE (Invitrogen) gels (2 h, 100 V). Gels were transferred to a Immobilon-FL PVDF Membranes (Millipore) using the Mini Blot Module western blotting system (Invitrogen) for 1 h in transfer buffer (192 mM glycine, 25 mM Tris, 20% methanol [v/v], 4 °C). Membranes were dried for 15 min in the fume hood, rehydrated with a 15 s methanol wash, and submerged in TBST (500 mM NaCl, 20 mM Tris pH 7.4, 0.05% Tween 20 [v/v]). Membranes were blocked in 5% nonfat dry milk (NFDM) in TBST for 1 h on a rocker at RT. Membranes were incubated with primary antibodies diluted (Table S2) in 1% NFDM in TBST with 0.02% (w/v) sodium azide overnight on a rocker at 4 °C. After the primary incubation, membranes were washed in TBST (3×, 5 min, RT) on a rocker. Membranes were incubated on a rocker with fluorophore-conjugated secondary antibodies diluted (Table S2) in 5% NFDM in TBST (1 h, RT). After the secondary incubation, membranes were washed in TBST (3×, 5 min, RT) on a rocker and imaged on LI-COR Odyssey CLx using the Image Studio v5.2 software (LI-COR). Special care was made to ensure that membranes were exposed to the light as little as possible to protect the fluorescent signal of the secondary antibody.