Journal: Frontiers in Cell and Developmental Biology
Article Title: STAT3 Localizes in Mitochondria-Associated ER Membranes Instead of in Mitochondria
Figure Lengend Snippet: Re-examining the existence and function of ‘mitochondrial STAT3.’ (A) Purification of mitochondria by sonication. (B) Purification of mitochondria by trypsinization. (C) Purification of mitochondria by washing with high concentration of salt combined with trypsinization. GRP78 was used as the ER marker; ATP5A, NDUFA9, NDUFA13, and VDAC were used as the mitochondrial marker; GAPDH was used as the cytosolic marker. (D) Sucrose density centrifuge of digitonin-solubilized mitochondria followed by Western blot analysis of STAT3 and mitochondrial complexes protein. (E) Co-immunoprecipitation experiment in digitonin-solubilized crude mitochondria. (F) ChIP-qPCR detection of STAT3-binding on mitochondrial DNA in mouse embryonic stem cells. (G) Serum reintroduction experiment in Neuro2A cells. (H) Quantification of Western blot results (G) in three independent experiments. * p
Article Snippet: Samples were then transferred to PVDF membrane (Thermo) and immunoblotted using anti-NDUFA9 (Invitrogen), anti-NDUFS3 (Invitrogen), anti-NDUFA13 (Invitrogen), anti-ATP5A (Invitrogen), anti-SDHA (CST), anti-VDAC (CST), anti-HSP60 (CST), anti-PHB1 (CST), anti-PDH (CST), anti-GAPDH (Sigma), anti-GRP78 (SCBT), anti-ABCA1 (SCBT), anti-FLOT1 (CST), anti-STAT3 (CST), anti-STAT1 (SCBT), anti-AMPK (CST), anti-ERK1/2 (CST), anti-p38 (CST), anti-SYP (Abcam), anti-ACSL4 (Abcam), all diluted in 5% BSA:TBST at 1:1000, followed by appropriate HRP-conjugated secondary antibodies (Thermo) incubation (diluted in 5% non-fat milk:TBST at 1:10,000), and developed using the SuperSignalTM West Femto Maximum Sensitivity Substrate (Thermo).
Techniques: Purification, Sonication, Concentration Assay, Marker, Western Blot, Immunoprecipitation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay