Rapid extraction of the proteasome pools from snap-frozen WT and PiZ mouse liver fragments.

<p><u>(A). Pilot extractions with increasing KCl concentrations in the absence and presence of Triton X-100</u>. A total of 17 extracts, 40 µl each, were prepared sequentially from a single 20 mg WT or PiZ mouse liver fragment, as indicated and described in text. Equal fraction (5 µl) of each extract was separated by 12.5% SDS-PAGE and analyzed by Western blot (WB) for the 20S subunits alpha, or by Commassie blue (CB) for total proteins. Samples shown in lanes 1–8 and 9–17 were analyzed on separate mini-gels that were processed side-by-side and exposed on a single large X-ray film. <u>(B). Extractions with 50 mM KCl in the absence and presence of Triton X-100. </u>Experiment like in A, except that 9 sequential extracts, 40 µl each, were prepared from a single 20 mg liver fragment using buffers that contained 50 mM KCl and either no detergent, 0.5% of Triton X-100, or 2% SDS, as indicated. Asterisks marks a space without sample loaded that was removed from the original 19S Rpt4 WB image to match the loading used in the 20S β5 WB. <u>(C). The origin of the proteasome pools</u>. Experiment like in B, except that with focus on proteins known to reside in specific subcellular locations: GAPDH and NFκB (cytosol); Cytochrome C (mitochondria); LC3 (autophagic vesicles); Calnexin and BiP (ER); Lamins A and C, and Histone H3 (nucleus). The total protein contents in WT and PiZ extracts were verified by Ponceau stain to be similar to those observed in Fig. 1A, CB (data not shown). Asterisk indicates where a lane without sample loaded (empty space) was removed from the original WB image, to match the arrangement of samples in other blots. The data are representative of four WT and four PiZ mice, with 3 randomly selected fragments extracted from each liver (24 extraction sets total).