Nap blocker

An equivalent amount of each subcellular fraction was mixed with 2x Laemmli sample buffer and heated in a boiling water bath for 10 minutes prior to loading cell equivalents on a 10% SDS-PAGE gel. Gels were run at 125 V until the dye front reached the bottom of the gel and then proteins were transferred to a nitrocellulose membrane at 25 V for 1.5 hours. Membranes were blocked with NAP-blocker (G-Biosciences) diluted 1:2 with Tris-buffered saline containing 0.1% Tween-20 (TBST) and then cut to allow for simultaneous probing with each antibody. Primary antibodies against Ply at 1:1000 (Statens Serum Institut) and CodY at 1:1500 (a gift from A.L. Sonenshein) were diluted accordingly in NAP-blocker mixed 1:4 with TBST and applied to each membrane at room temperature for 1 hour with rocking. Membranes were washed three times for 5 minutes each with TBST. Appropriate Cy5-conjugated secondary antibody at 1:1000 (Invitrogen, Inc.) was applied to each membrane as described above for the primary and then each blot was washed as described above. Membranes were scanned with a Fuji FLA-9000 instrument and the amount of fluorescence was quantitated using MultiGauge analysis software (Fujifilm, Corp.)

The cell grown in 6‐well plates and treated with indicated condition described in figure legends section was washed twice with PBS, followed by preparing cell lysates using RIPA lysis buffer. The total proteins were mixed with electrophoresis sample buffer (Cat. No. #161‐0747; Bio‐Rad Laboratories, Inc), boiled and run on SDS‐PAGE, followed by transferred to polyvinylidene fluoride membranes. The blots were then blocked with NAP‐Blocker (G‐Biosciences). The blots were probed with primary antibodies (1:1000) for overnight at 4°C and subsequently incubated with HRP–conjugated secondary antibody (1:2000) against rabbit or mouse IgG for 1 hour at room temperature. The relative information of antibodies used in Western blot was provided in Materials section. The indicated proteins were visualized using ECL^™ Prime Western Blotting System (Cat. No. RPN2232; GE Healthcare), detected using ImageQuant LAS 4000 bimolecular imager (GE Healthcare Life Sciences). Quantitative analysis of Western blot was conducted using a computing densitometer and ImageQuant (Molecular Dynamics).