Journal: Febs Letters

Article Title: Mitochondrial intermediate peptidase is a novel regulator of sirtuin‐3 activation by caloric restriction

doi: 10.1002/1873-3468.12914

Figure Lengend Snippet: Maturation of SIRT 3 was dependent on MIPEP . (A) Protein levels of SIRT 3 in mock or FLAG ‐Sirt3 expressing 3T3‐L1 cells were obtained by immunoblotting with high‐density gel (15%). Cells were treated with or without 10 μ m MG 132 for 6, 12, 24 and 48 h. β‐ ACTIN was used as a loading control. (B) Protein levels of MIPEP , SIRT 3, COX 4 and MDH 2 in sh Mipep 3T3‐L1 cells were obtained by immunoblotting. GAPDH was used as a loading control. The lower panel is the quantitative data. (C) mRNA levels of Sirt3 , Cox4 and Mdh2 in sh Mipep 3T3‐L1 cells were obtained by real‐time RT ‐ PCR . Rps18 was used as an internal control. (D) Expression levels of Ac‐Mn SOD (K122 acetylated Mn SOD ) and total Mn SOD proteins in sh Mipep 3T3‐L1 cells were obtained by immunoblotting. α‐ TUBULIN was used as a loading control. Values shown in all panels are means ± SEM ( n = 4–6). * P < 0.05, ** P < 0.01, analysed by Student's t ‐test.

Article Snippet: Primary antibodies against SIRT3 (Cell Signaling Technology (CST), Beverly, MA, USA), MIPEP (Santa Cruz Biotechnology, San Francisco, CA, USA), COX4 (CST), MDH2 (CST), manganese superoxide dismutase (MnSOD) (CST), MnSOD (acetyl K122) (Abcam, Cambridge, UK), FLAG (Sigma), β‐ACTIN (Sigma), α‐TUBULIN (Sigma) and GAPDH (Santa Cruz) were used.

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR