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. 2012 Oct 5;91(4):729–736. doi: 10.1016/j.ajhg.2012.08.019

Figure 6.

Figure 6

RMND1 Localization in Mammalian Cells by Immunohistochemistry and Quantitation by Immunoblot Analysis

(A) RMND1 cDNA was amplified from a commercial vector containing the human full-length cDNA with the use of a BamHI-recognition-site-integrated forward primer 5′-AGG ATCCGCCATGCCAGCCACACTCCTCAGAGCCG-3′ and AgeI-recognition-site-integrated reverse primer 5′-CGACCGGTGATTTCATGGTTGGAAGGTGTG-3′. PCR product was digested with BamHI and AgeI and cloned into a pEYFP expression vector in-frame and upstream of the YFP coding sequence. Fidelity of the fusion RMND1-YFP construct in the expression vector was verified by sequencing, and 2.5 μg was used for transiently transfecting human embryonic kidney (HEK) 293T cells as described above. Culture media were supplemented with 0.1 μM MitoTracker Red (Invitrogen) for 30 min before the cells were fixed with 4% formalin PBS for the detection of mitochondria.

(B) Purification of mitochondria and cellular fractions was performed as previously described,27 and immunoblot analysis was performed with primary antibodies anti-RMND1 (1:1,000, Sigma), anti-Complex II (1:5,000, MitoScience, Abcam, Cambridge, MA), anti-APH1a (1:1,000, Abcam), and anti-Vinculin (1:5,000, Sigma-Aldrich, St. Louis, MO) and either peroxidase-conjugated anti-rabbit (1:2,000) or peroxidase-conjugated anti-mouse IgG (1:5,000) as a secondary antibody (Sigma-Aldrich). Cellular fractions were isolated from HeLa cells (25 μg of protein from the total lysate [L]), and equal amounts (6 μg) from each fraction (cytoplasmic fraction [CF], endoplasmatic reticulum [ER], crude mitochondria [CMt], pure mitochondria [PMt], and ER + mitochondria [ERMt]) were loaded onto the gel. The known molecular masses (in kDa) of proteins used for confirming cell-fraction enrichment are indicated in the left margin of the gel.

(C) Immunoblot analysis of total HEK 293T cell extracts transfected with YFP-RMND1 fusion protein with the antibody against green fluorescent protein confirmed the specificity of the two bands detected by the RMND1 antibody.