Actin nb600 501

Cells were lysed with RIPA buffer (10 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% TritonX-100, 1% sodium deoxycholate) containing a cocktail of protease inhibitors (Roche). Equivalent amounts of proteins determined by the DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA) were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Hybond-C Super; Amersham, Buckinghamshire, UK). Nonspecific antibody binding sites were blocked with skim milk in phosphate-buffered saline (PBS) with 0.1% Tween 20 (PBST), then reacted with primary antibodies for HADHα (sc-82185), HADH (sc-55661 and sc-271496) and α Tubulin (sc-5546) from Santa Cruz Biotechnology; actin (NB600-501; Novus Biologicals, Littleton, CO, USA); GAPDH (GTX100118) and TOM70 (GTX85353) from GeneTex (Irvine, CA, USA); Cytochrome C (#556433; BD, Franklin Lakes, New Jersey, USA); Calreticulin (#2891), phospho-STAT1 (Tyr701) (#9171), STAT1 (#9172), phospho-AMPKα (Thr172) (#2535), and AMPKα (#2532) from Cell Signaling Technology(Danvers, MA, USA); GFP (#11814460001; Roche); V5-tag (V8012) and Flag-tag (F7425) from Sigma; or HA-tag (MMS-101R; Covance, Princeton, New Jersey, USA), and then incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (Amersham). Signals were detected by enhanced chemiluminescence (Amersham).

Protein samples were prepared by using RIPA lysis buffer (10 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% TritonX-100, 1% sodium deoxycholate) containing protease and phosphatase inhibitors (Roche). After SDS-PAGE separation, samples were transferred to a nitrocellulose membrane (Hybond-C Super, Amersham), which was blocked with skim milk in phosphate buffered saline (PBS) with 0.1% Tween-20 (PBS-T), then reacted with the primary antibodies for caspase-3 (#9662) and phospho-IRF3 (S396) (#4961; both Cell Signaling); IRF3 (sc-9082), MFN1 (sc-50330), and MFN2 (sc-100560; all Santa Cruz Biotechnology); OPA1 (GTX48589), and Fis1 (GTX111010; both GeneTex); actin (NB600-501; Novus Biologicals); HA (#3724), myc (#2278; both Cell Signaling); V5 (R960-25; Life Technologies); and Flag M2 (F1804; Sigma-Aldrich). Blots were treated with horseradish peroxidase-conjugated secondary antibody (111-035-144 and 115-035-146; Jackson ImmunoResearch), and signals were detected by enhanced chemiluminescence (Pierce). The relative ratios of band density were quantified by ImageJ.