Journal: Antioxidants & Redox Signaling
Article Title: Identification of a Redox-Modulatory Interaction Between Uncoupling Protein 3 and Thioredoxin 2 in the Mitochondrial Intermembrane Space
Figure Lengend Snippet: Submitochondrial localization of the UCP3/Trx2 interaction. (A) Mitochondria isolated from HeLa cells transfected with UCP3 and Trx2 were suspended in RIPA buffer+SDS or lysis buffer+Triton X-100, respectively. Mitochondrial pellets and supernatants were subjected to SDS-PAGE followed by immunoblotting (IB) with anti-myc, anti-V5, anti-COX4, and anti-DNA polymerase γ (DPγ) antibodies. PPT, mitochondrial pellet after centrifugation; SDS, RIPA buffer; SUP, supernatant after centrifugation; Triton X-100; lysis buffer. (B) . Mitochondrial pellets and supernatants were resuspended in SDS-PAGE buffer, and then loaded onto an SDS-PAGE gel. Immunoblots show the presence of the intermembrane space (IMS) resident Smac and the matrix resident HSP60 in mitochondria that were untreated or treated with proteinase K (Pro K) (lanes 2–7) and increasing concentrations of digitonin (DIG) (lanes 3–7, 0.1 to 0.7 mg/ml DIG). (C) N (VN)- and C (VC)- terminal fragments of Venus fluorescent proteins were fused to the C-terminus of UCP3 (resides 1–308) (UCP3-VN) or ΔCUCP3 (residues1–234) (ΔCUCP3-VN), and the C-terminus of Trx2, respectively. The fragments of fluorescent proteins of belonging to UCP3-VN and ΔCUCP3-VN were localized to the mitochondrial IMS and matrix, respectively. (D) HeLa cells were transfected with UCP3-VN, Trx2-VC, ΔCUCP3-VN and Trx2-VC, and UCP3-VN and Trx2-VC. Cell extracts (100 μg) were immunoprecipitated (IP) with anti-myc antibodies and analyzed by IB to detect V5 (UCP3) and myc (Trx2). β-Actin was used as an internal standard. (E) Fluorescent images of HeLa cells transfected with UCP3-VN, Trx2-VC, ΔCUCP3-VN and Trx2-VC, and UCP3-VN and Trx2-VC as indicated in each panel. (F) ).
Article Snippet: The proteins were transferred to a nitrocellulose membrane and probed with primary antibody according to the manufacturer's instructions; anti-V5, anti-UCP3, anti-aconitase 2, anti-β-actin (Abcam, Cambridge, MA), anti-cytochrome c (BD Biosciences, Franklin Lakes, NJ), anti-myc, anti-phospho-p38, anti-p38 (Cell Signaling), anti-COX4 (Clontech, Mountain View, CA), anti-DNA polymerase γ (Neo Markers, Fremont, CA), anti-T7 (Novagen, Gibbstown, NJ), and anti-thioredoxin 2 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were used.
Techniques: Isolation, Transfection, Lysis, SDS Page, Centrifugation, Western Blot, Immunoprecipitation