Ib24001

The protein concentrations of the cellular fractions were measured using colorimetric RC DC assay kit (#5000122, Bio-Rad). 15 μg of protein per sample (except for mitochondria fractions, that were too dilute to take 15 μg, from which maximum volume of sample was run) was run on a Bis-Tris 4–12% gel (NP0321, NuPage, Invitrogen, MA) with MOPS Buffer (200V, 45 min) and transferred to a 0.2 micron PVDF membrane (IB24001, Invitrogen) with iBlot2 (Invitrogen). Immediately after the transfer the blot was stained for total proteins (Total Stain Q, AC2225, Azure Biosystems), dried, and imaged with a Sapphire Biomolecular Imager (488 nm laser, Azure Biosystems). After imaging the total protein stain the membranes were re-hydrated with methanol, washed with distilled water followed by PBS, and blocked with blocking buffer (cat# MB070, Rockland, PA) for 1 h at room temperature (RT) and then probed with primary antibodies at 4°C overnight in blocking buffer. The following day the blots were washed and probed with secondary antibodies. The blots were scanned with Odyssey scanner (LI-COR Biosciences, Lincoln, NE). The optical density of each protein band was normalized to the optical density of the total proteins of the corresponding sample lane. Optical densities were measure using ImageJ (version 1.53f, imagej.nih.gov). All the used antibodies are listed in Table 2.