Anti egfp b 2

The rat full-length MECR (GenBank accession no. AB015724), cytosolic variant of MECR, and PPARα (GenBank accession no. NM_001001928) were cloned using the Gateway Cloning System (Invitrogen) as previously described [14 (link)]. After amplification of the three genes, PCR products were cloned into the pDONR207 vector and then subcloned into self-constructed destination vectors such as pDS_XB-HA, pDs_XB-Flag, pDS_XB-enhanced green fluorescent protein (EGFP), and pDS_XB-mCherry expression vectors. Anti-Flag (F6531) antibody was purchased from Sigma (St. Louis, MO, USA). Anti-EGFP (B-2) and antihemagglutinin (anti-HA) (F-7) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Immunoblot analysis was performed essentially as described.⁶ (link) Typically, 50 μg of protein lysate were used for immunoblot. Detection was done using enhanced Chemiluminescent reagent (Amersham) or Super signal West Dura (Pierce). Antibodies used in this study are: anti-p73 (ER15; 1:1000; Calbiochem), anti-p73 (GC15; 1:1000; Calbiochem), anti-FLAG (M2; 1:2000; Stratagene), anti-p27 (C-19; 1:500; Santa Cruz Biotechnology), anti-Actin (AC-74, 1:5000; Sigma-Aldrich), anti-Egfp (B-2; 1:2000; Santa Cruz Biotechnology), anti-HIF-1α (1:250; BD Biosciences).