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. 2004 Jun 17;23(13):2520–2530. doi: 10.1038/sj.emboj.7600271

Figure 2.

Figure 2

Mmr1p localization. (A) (a–e) MMR1HA cells (a–e, strain yTO053) were grown to mid-log phase, fixed, and stained using anti-HA antibodies visualized by FITC (a and c, green) and with DAPI for DNA (b and d, red). (e) Merging (c) and (d). (f–h) MMR1HA cells with mitochondria-targeted GFP were grown to mid-log phase, fixed, and stained using anti-HA antibodies visualized with Alexa 546. (f) Mitochondria (green); (g) Mmr1pHA (red); and (h) merged. Scale bar: 5 μm. (B) Cell fractionation. Lysate of MMR1HA cells was fractionated by differential centrifugation. Equal amounts of cell equivalents of each fraction were analyzed by Western blotting. Total: total cell lysate; P13: the P13 pellet fraction; P100: the P100 pellet fraction; S100: the S100 supernatant fraction. Pgk1p, 3-phosphoglycerate kinase (a cytosolic marker), was enriched in the S100 fraction. (C) Fractionation of P13 fraction. P13 fraction (lane 1) from MMR1HA cells was loaded on top of a three-step sucrose gradient (60, 32, 23, and 15% sucrose) and centrifuged. Equal amounts of cell equivalents of each fraction (fraction from 32/60% interface as purified mitochondrial fraction (lane 2) and fraction from 15 and 23% phases as residual membrane fraction (lane 3)) were analyzed by Western blotting. (D) Treatment of salt and alkali. Cell lysates from MMR1HA cells without (NT) or with 0.5 M NaCl (salt) or 0.1 M sodium carbonate (alkali) were centrifuged to separate soluble fraction (S) from pellet fraction (P), which were analyzed by Western blotting. (E) Proteinase K treatment. Cell lysates from MMR1HA cells, without (lane 1) or with (lane 2) proteinase K treatment, were centrifuged and the resultant pellets were analyzed by Western blotting.