Tim 44

rBA were cultured in 12-well plates, differentiated and infected as described above. Cells were harvested in lysis buffer (RIPA), and protein concentration was determined using a BCA protein assay kit (Thermoscientific). Samples were separated on 8% and 12% SDS-PAGE gels, for CPT1A and UCP1 respectively, and then transferred onto PVDF membranes (Millipore). The following primary antibodies were used: CPT1A (1/6,000) [24 (link)], Tim 44 (1/5,000; BD Bioscience), UCP1 (1/1,000; Abcam) and b-actin (I-19; 1/4,000; Santa Cruz). Blots were incubated with the appropriate IgG-HRP-conjugated secondary antibody. Protein bands were visualized using the ECL immunoblotting detection system (GE Healthcare) and developed on an ImageQuant LAS4000 mini Fuji luminescence imagining system. For the analysis of protein expression, bands from at least three independent experiments were quantified by densitometry using Image J analysis software.

Frozen tissue was homogenized in protein extraction buffer (30 mM Hepes, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 0.5% sodium deoxycholate [DOC] with phosphatase inhibitors and protease inhibitors). Protein concentration was determined using a BCA protein assay kit (Thermoscientific). Samples were separated on 8% and 12% SDS-PAGE gels and then transferred onto PVDF membranes (Millipore). The following primary antibodies were used: TIM44 (1/5,000; BD Bioscience), UCP1 (1/1,000; Abeam), mitochondrial complexes I, II, III, IV and V (1/5,000; Life Technologies), α-tubulin (1/5,000; Abcam) and β-actin (1/50,000; Sigma-Aldrich). Blots were incubated with the appropriate IgG-HRP-conjugated secondary antibody. Protein bands were visualized using the ECL immunoblotting detection system (GE Healthcare) and developed on an ImageQuant LAS4000 mini Fuji luminescence imagining system. For the analysis of protein expression, bands from at least three independent experiments were quantified by densitometry using Image J analysis software.

Approximately 40 μg of proteins were separated on 10 or 12% SDS-PAGE Laemmli gels, depending on the molecular weight of the protein of interest. A wet-blotting system (Bio Rad) was used for protein transfer to nitrocellulose membrane, which were stained with 0.1% Ponceau (in 5% acetic acid) to evaluate transfer efficiency. Membranes were blocked for 1 h at RT in 5% non-fatty milk (in PBS). Primary antibodies were diluted in PBS with 5% BSA and 0.1% Tween 20. The following antibodies were used at a concentration of 1:1000: p38α (Cell Signaling, 9218), P-p38 (Thr180/Tyr182) (Cell Signaling, 9211 S), P-MAPKAPK2 (Thr222) (Cell Signaling, 3044 S), P-Hsp27 (Ser82) (StressGene, SPA-523), P-PDHE1-A type I (Ser293) (Merk Millipore, ABS204), c-Myc (Abcam, ab32072), Tim-44 (BD, T14720), TFAM (Abcam, ab131607), VDAC-1 (Abcam, ab14734), P-CREB (Ser133) (Cell Signaling, #9191), P-eIF4E (Ser209) (Cell Signaling, #9741). The MKK6 rabbit antiserum has been previously described^{46 (link)}, and the p38γ antibody was kindly provided by Ana Cuenda (CNB, Madrid). Tubulin (Sigma, T9026) was used at 1:5000 as a loading control. Secondary Alexa Fluor-conjugated antibodies were diluted 1:5000 in 1% non-fatty milk. Membranes were analysed using the Odyssey Infrared Imaging System (Li-Cor Biosciences).