Journal: PLoS ONE

Article Title: Upregulation of UCP2 by Adiponectin: The Involvement of Mitochondrial Superoxide and hnRNP K

doi: 10.1371/journal.pone.0032349

Figure Lengend Snippet: A , PCs and NPCs were isolated from the liver tissues of C57 and AKO mice. The protein (left and middle panel) and mRNA (right panel) expression of UCP2 was measured by Western blotting and QPCR, respectively. SSBP-1 was used as the protein loading control and β-actin as the internal control for quantifying gene expressions. QPCR results were plotted as fold changes against the C57 PC samples. *, P <0.05 vs C57 NPC samples, n = 3. B , AKO mice were treated with adenoviruses encoding luciferase (Luci) or adiponectin (ADN). UCP2 protein (left and middle panel) and mRNA (right panel) levels in PCs and NPCs were analyzed as above. QPCR results were presented as fold changes against the Luci PC controls. *, P <0.05 vs corresponding controls, n = 3.

Article Snippet: Protein lysates were heated at 95°C for 5 min, separated by SDS-PAGE, and transferred to PVDF membrane for immunoblotting with the specific antibodies against UCP2 (R&D Systems, #AF4739), single-strand binding protein-1 (SSBP-1, Santa Cruz, #sc-34727), liver sinusoidal endothelial cell marker SE-1 (Novus Biologicals, NB110-68095), F4/80 (Abcam, #ab6640), hnRNP K (Santa Cruz, #sc-25373) or β-actin (Sigma, #A5316).

Techniques: Isolation, Expressing, Western Blot, Control, Luciferase

Journal: PLoS ONE

Article Title: Upregulation of UCP2 by Adiponectin: The Involvement of Mitochondrial Superoxide and hnRNP K

doi: 10.1371/journal.pone.0032349

Figure Lengend Snippet: PBS or 50 µg of murine adiponectin protein was injected into the portal vein of AKO mice livers. Liver tissues were collected for evaluation of UCP2 expression. The protein abundance in mitochondria (A), mRNA expression in total tissue lysates (B), and the protein content in PC and NPC fractions (C) were analyzed as in . *, P <0.05 vs vehicle treated samples, n = 3.

Article Snippet: Protein lysates were heated at 95°C for 5 min, separated by SDS-PAGE, and transferred to PVDF membrane for immunoblotting with the specific antibodies against UCP2 (R&D Systems, #AF4739), single-strand binding protein-1 (SSBP-1, Santa Cruz, #sc-34727), liver sinusoidal endothelial cell marker SE-1 (Novus Biologicals, NB110-68095), F4/80 (Abcam, #ab6640), hnRNP K (Santa Cruz, #sc-25373) or β-actin (Sigma, #A5316).

Techniques: Injection, Expressing, Quantitative Proteomics

Journal: PLoS ONE

Article Title: Upregulation of UCP2 by Adiponectin: The Involvement of Mitochondrial Superoxide and hnRNP K

doi: 10.1371/journal.pone.0032349

Figure Lengend Snippet: AKO mice were treated as in . The NPCs were used for further fractionation to collect those enriched with Kupffer (K)- and sinusoidal endothelial (E) cells. The enrichment of the two cell types were confirmed by Western blotting using macrophage marker F4/80 and sinusoidal endothelial marker SE-1, respectively (A). UCP2 expression was monitored as in . After densitometry analysis, the protein ratio of UCP2/β-actin was calculated and presented as fold changes against Luci Kupffer samples (B). UCP2 gene expression was also quantified in four types of cells treated with or without adiponectin (10 µg/ml) (C). *, P <0.05 and **, P <0.01 vs corresponding controls, n = 3.

Article Snippet: Protein lysates were heated at 95°C for 5 min, separated by SDS-PAGE, and transferred to PVDF membrane for immunoblotting with the specific antibodies against UCP2 (R&D Systems, #AF4739), single-strand binding protein-1 (SSBP-1, Santa Cruz, #sc-34727), liver sinusoidal endothelial cell marker SE-1 (Novus Biologicals, NB110-68095), F4/80 (Abcam, #ab6640), hnRNP K (Santa Cruz, #sc-25373) or β-actin (Sigma, #A5316).

Techniques: Fractionation, Western Blot, Marker, Expressing, Gene Expression

Journal: PLoS ONE

Article Title: Upregulation of UCP2 by Adiponectin: The Involvement of Mitochondrial Superoxide and hnRNP K

doi: 10.1371/journal.pone.0032349

Figure Lengend Snippet: Two inhibitors, actinomycin D (ActD, A, B and C) or cycloheximide (CHX, A, B and D), were administered together with or without adiponectin protein into liver tissues of AKO mice livers. The protein (A) and mRNA (B) abundance of UCP2 was monitored by Western blotting and QPCR, respectively. The relative protein expression was also monitored in PCs/NPCs lysates (C and D). *, P <0.05 vs corresponding controls, n = 3.

Article Snippet: Protein lysates were heated at 95°C for 5 min, separated by SDS-PAGE, and transferred to PVDF membrane for immunoblotting with the specific antibodies against UCP2 (R&D Systems, #AF4739), single-strand binding protein-1 (SSBP-1, Santa Cruz, #sc-34727), liver sinusoidal endothelial cell marker SE-1 (Novus Biologicals, NB110-68095), F4/80 (Abcam, #ab6640), hnRNP K (Santa Cruz, #sc-25373) or β-actin (Sigma, #A5316).

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Upregulation of UCP2 by Adiponectin: The Involvement of Mitochondrial Superoxide and hnRNP K

doi: 10.1371/journal.pone.0032349

Figure Lengend Snippet: Mitochondrial respiration chain inhibitors, including rotenone (Rot), sodium azide (NaN 3 ) and antimycin A (AntA) were administered into liver tissues of AKO mice as described in . The effects of each inhibitor on adiponectin-induced UCP2 protein expression in mitochondria of liver tissues (A), UCP2 mRNA expression in total tissue lysates (B) were evaluated. The mitochondria protein content of UCP2 (C) and hnRNPK (D) in PCs and NPCs were compared by Western blotting. *, P <0.05 vs corresponding controls, n = 3.

Article Snippet: Protein lysates were heated at 95°C for 5 min, separated by SDS-PAGE, and transferred to PVDF membrane for immunoblotting with the specific antibodies against UCP2 (R&D Systems, #AF4739), single-strand binding protein-1 (SSBP-1, Santa Cruz, #sc-34727), liver sinusoidal endothelial cell marker SE-1 (Novus Biologicals, NB110-68095), F4/80 (Abcam, #ab6640), hnRNP K (Santa Cruz, #sc-25373) or β-actin (Sigma, #A5316).

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Upregulation of UCP2 by Adiponectin: The Involvement of Mitochondrial Superoxide and hnRNP K

doi: 10.1371/journal.pone.0032349

Figure Lengend Snippet: A schematic summary: Adiponectin-evoked transient elevation of mitochondrial O 2• − serves as a trigger for the translocation hnRNP K, which subsequently promotes the stabilization of UCP2 mRNA and its protein synthesis in mitochondria of hepatic endothelial cells.

Article Snippet: Protein lysates were heated at 95°C for 5 min, separated by SDS-PAGE, and transferred to PVDF membrane for immunoblotting with the specific antibodies against UCP2 (R&D Systems, #AF4739), single-strand binding protein-1 (SSBP-1, Santa Cruz, #sc-34727), liver sinusoidal endothelial cell marker SE-1 (Novus Biologicals, NB110-68095), F4/80 (Abcam, #ab6640), hnRNP K (Santa Cruz, #sc-25373) or β-actin (Sigma, #A5316).

Techniques: Translocation Assay

Journal: Endocrinology

Article Title: Follistatin Targets Distinct Pathways To Promote Brown Adipocyte Characteristics in Brown and White Adipose Tissues

doi: 10.1210/en.2016-1607

Figure Lengend Snippet: List of Antibodies

Article Snippet: After the treatment with enhanced chemiluminescence reagent (Pierce; Thermo Scientific), the membranes were exposed to an X-ray film (Kodak; Sigma Aldrich, St. Louis, MO) and Image by Image Quant (GE Lifescience, Pittsburgh, PA) software for densitometric analysis ( 28, 32 ). table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Peptide/Protein Target Name of Antibody Manufacturer, Catalog Number Antibody RRID Species Dilution Used UCP1 Uncoupling protein1 antibody Abcam ab10983 AB_2241462 Rabbit polyclonal 1:1000 UCP2 Uncoupling protein2 antibody Novus Biologicals NB100-78377 AB_1085929 Rabbit polyclonal 1:1000 UCP3 Uncoupling protein3 antibody Abcam ab3477 AB_2304253 Rabbit polyclonal 1:1000 PRDM16 PRDM16 antibody (N16) Santa Cruz Biotech sc-130243 AB_2284344 Rabbit polyclonal 1:500 PGC-1 α PGC-1 α antibody (H-300) Santa Cruz Biotech sc-13067 AB_2166218 Rabbit polyclonal 1:1000 Adiponectin Adiponectin antibody R & D Systems MAB1119 AB_2305045 Rat monoclonal 1:1000 AMPK α AMPK α antibody Cell Signaling 2532 AB_330331 Rabbit monoclonal 1:1000 pAMPK α pAMPK α antibody Cell Signaling 2535 AB_331250 Rabbit monoclonal 1:1000 Glut4 Glucose transporter 4 antibody Abcam ab65267 AB_1140009 Mouse monoclonal 1:1000 p38MAPK p38MAPK antibody Cell Signaling 9212S AB_330713 Rabbit polyclonal 1:1000 pp38MAPK pp38MAPK antibody Cell Signaling 9211S AB_331641 Rabbit polyclonal 1:1000 ERK1/2 ERK1/2 antibody Cell Signaling 9102S AB_330744 Rabbit polyclonal 1:1000 pERK1/2 pERK1/2 antibody Cell Signaling 9101S AB_331646 Rabbit polyclonal 1:1000 Myf5 Myf5 antibody Santa Cruz Biotech sc-302 AB_631994 Rabbit polyclonal 1:1000 Cd137 Cd137 antibody Abcam ab3169 AB_303572 Mouse monoclonal 1:1000 BMP7 BMP7 antibody Abcam ab54904 AB_940598 Mouse monoclonal 1:1000 Eva1 Eva1 antibody Novus Biologicals NBP1-88937 AB_11016118 Rabbit polyclonal 1:1000 β -actin Beta actin antibody Santa Cruz Biotech sc-81178 AB_2223230 Mouse monoclonal 1:5000 GAPDH GAPDH antibody Millipore MAB374 AB_2107445 Mouse monoclonal 1:5000 pSmad2/3 pSmad2/3 antibody Cell Signaling 8828 AB_2631089 Rabbit monoclonal 1:1000 AcvRIIB AcvRIIB antibody Abcam ab76940 AB_1565807 Mouse monoclonal 1:1000 Smad3 Smad3 antibody Cell Signaling 9513 AB_2286450 Rabbit polyclonal 1:1000 Open in a separate window List of Antibodies

Techniques:

Journal: Journal of Cell Science

Article Title: Degradation of an intramitochondrial protein by the cytosolic proteasome

doi: 10.1242/jcs.060004

Figure Lengend Snippet: Proteasome inhibitors block UCP2 degradation in cells. INS-1E cells were preincubated with proteasome inhibitors for 2 hours then treated with 10 μg/ml cycloheximide. Samples were taken at the times shown, separated by SDS-PAGE (1×105 cells/lane) and immunoblotted for UCP2. (A) UCP2 degradation in cells treated with proteasome inhibitor cocktail-1 [PIC-1; containing 10 μM MG132, 10 μM lactacystin (Lact.) and 30 μM PI-1] or proteasome inhibitor cocktail-2 [PIC-2; containing 30 μM ALLN, 5 μM clastolactacystin β-lactone (Clasto.) and 5 μM epoxomicin (Epoxo.)] (n=6). (B-D) UCP2 degradation in cells treated with: (B) 10 μM MG132, 10 μM lactacystin or 30 μM PI-1 (n=3); (C) 30 μM ALLN, 5 μM clastolactacystin β-lactone or 5 μM epoxomicin (n=3); or (D) 5 μM adamantane-acetyl-6-aminohexanoyl-3-leucinyl-3-vinyl-methylsulfone (AdaAhx3L3VS; Ada.; n=3). Values are means ± s.e.m., corrected for loading (β-actin). Statistical significance was determined by repeated measures ANOVA (comparison of matching non-zero time points) with Dunnett's post-hoc testing (*P<0.05, **P<0.01, ***P<0.001). Typical UCP2 immunoblots (molecular mass ~30 kDa) are shown in supplementary material Fig. S1.

Article Snippet: Immunoblotting Typically, 1×10 5 cells or 25 μg mitochondrial protein in gel loading buffer (10% (w/v) SDS, 250 mM Tris-HCl (pH 6.8), 5 mM EDTA, 50% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 0.05% (w/v) Bromophenol Blue) were separated by 12.5% SDS-PAGE, transferred onto a Protran nitrocellulose membrane (Whatman) using the semi-dry method (20 V for 30 minutes), and probed with 0.2 μg/ml anti-human UCP2 goat polyclonal IgG antibody (Santa Cruz Biotechnology, cat. no. sc-6525), 0.2 μg/ml anti-human adenine nucleotide translocase (ANT) goat polyclonal IgG antibody (Santa Cruz Biotechnology, cat. no. sc-9300), 0.2 μg/ml anti-human β-actin rabbit polyclonal IgG antibody (Abcam, UK, cat. no. ab8227), or 0.2 μg/ml anti-HA (Abcam, UK, cat. no. ab9110).

Techniques: Blocking Assay, SDS Page, Comparison, Western Blot

Journal: Journal of Cell Science

Article Title: Degradation of an intramitochondrial protein by the cytosolic proteasome

doi: 10.1242/jcs.060004

Figure Lengend Snippet: Ubiquitin involvement in UCP2 degradation. (A) INS-1E cells were treated with 200 nM scrambled (Scr) or UCP2 (KD) siRNA for 48 hours. Cells were lysed with IP buffer and lysates were incubated for 1 hour with protein-A-conjugated beads and anti-UCP2 or anti-Ub antibodies, or non-antigen-specific (normal) immunoglobulin (N.Ig) at 4°C then pelleted and denatured by boiling in gel loading buffer. Proteins were separated by SDS-PAGE and immunoblotted for Ub, UCP2 or ANT. Non-specific: bands not dependent on specific interactions with UCP2 or ubiquitin. (B) INS-1E cells were transfected with 200 nM scrambled siRNA (Scr) or siRNAs targeted against UCP2 (KD). Scr/UCP2-KD cells used in the immunoprecipitation experiments were immunoblotted for UCP2, ANT and β-actin. Cell loading: 1×105 cells/lane. (C,D) Purified HA-tagged WT-ubiquitin, K48R-ubiquitin and lysine KO-ubiquitin plasmids were transfected at 0.5 μg/ml DNA for 20-24 hours. INS-1E cells were then treated with 10 μg/ml cycloheximide (CHX), harvested at the time points shown and resuspended in gel loading buffer. Proteins were separated using SDS-PAGE (1×105 cells/lane) and immunoblotted for HA (C) or UCP2 (D). Values in D are means ± s.e.m. (n=3), corrected for loading (β-actin). Statistical significance was determined by repeated measures ANOVA (comparison of matching non-zero time points) with Dunnett's post-hoc testing (*P<0.05, **P<0.01, ***P<0.001). Typical UCP2 immunoblots (molecular mass ~30 kDa) are shown.

Article Snippet: Immunoblotting Typically, 1×10 5 cells or 25 μg mitochondrial protein in gel loading buffer (10% (w/v) SDS, 250 mM Tris-HCl (pH 6.8), 5 mM EDTA, 50% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 0.05% (w/v) Bromophenol Blue) were separated by 12.5% SDS-PAGE, transferred onto a Protran nitrocellulose membrane (Whatman) using the semi-dry method (20 V for 30 minutes), and probed with 0.2 μg/ml anti-human UCP2 goat polyclonal IgG antibody (Santa Cruz Biotechnology, cat. no. sc-6525), 0.2 μg/ml anti-human adenine nucleotide translocase (ANT) goat polyclonal IgG antibody (Santa Cruz Biotechnology, cat. no. sc-9300), 0.2 μg/ml anti-human β-actin rabbit polyclonal IgG antibody (Abcam, UK, cat. no. ab8227), or 0.2 μg/ml anti-HA (Abcam, UK, cat. no. ab9110).

Techniques: Ubiquitin Proteomics, Incubation, SDS Page, Transfection, Immunoprecipitation, Purification, Comparison, Western Blot

Journal: Journal of Cell Science

Article Title: Degradation of an intramitochondrial protein by the cytosolic proteasome

doi: 10.1242/jcs.060004

Figure Lengend Snippet: Regulation of UCP2 degradation by mitochondrial bioenergetics. INS-1E cells were treated with the effectors described below for 30 minutes, then treated with 10 μg/ml cycloheximide. Samples were taken at the times shown, separated by SDS-PAGE (1×105 cells/lane), immunoblotted for UCP2 and quantified by densitometry. (A) Inhibition of respiratory chain using 10 μM rotenone (inhibits complex I), 10 μM antimycin A or 10 μM myxothiazol (inhibit Qo and Qi site of complex III, respectively). (B) Treatment with 20 μM FCCP (dissipates Δp), 0.5 μM nigericin (dissipates ΔpH) or 75 μM menadione. (C) Treatment with 1 μg/ml oligomycin (inhibits Fo/F1 ATP synthase) with or without 20 μM FCCP, or lysosomal inhibitor 10 mM NH4Cl. Values are means ± s.e.m. (n=3), corrected for loading (β-actin). Statistical significance was determined by repeated measures ANOVA (comparison of matching non-zero time points) with Dunnett's post-hoc testing (*P<0.05, **P<0.01, ***P<0.001). Typical UCP2 immunoblots (molecular mass ~30 kDa) are shown in supplementary material Fig. S2.

Article Snippet: Immunoblotting Typically, 1×10 5 cells or 25 μg mitochondrial protein in gel loading buffer (10% (w/v) SDS, 250 mM Tris-HCl (pH 6.8), 5 mM EDTA, 50% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 0.05% (w/v) Bromophenol Blue) were separated by 12.5% SDS-PAGE, transferred onto a Protran nitrocellulose membrane (Whatman) using the semi-dry method (20 V for 30 minutes), and probed with 0.2 μg/ml anti-human UCP2 goat polyclonal IgG antibody (Santa Cruz Biotechnology, cat. no. sc-6525), 0.2 μg/ml anti-human adenine nucleotide translocase (ANT) goat polyclonal IgG antibody (Santa Cruz Biotechnology, cat. no. sc-9300), 0.2 μg/ml anti-human β-actin rabbit polyclonal IgG antibody (Abcam, UK, cat. no. ab8227), or 0.2 μg/ml anti-HA (Abcam, UK, cat. no. ab9110).

Techniques: SDS Page, Inhibition, Comparison, Western Blot

Journal: Journal of Cell Science

Article Title: Degradation of an intramitochondrial protein by the cytosolic proteasome

doi: 10.1242/jcs.060004

Figure Lengend Snippet: UCP2 attachment and retrotranslocation. (A) INS-1E cells were treated with 200 nM scrambled siRNA or siRNAs targeted against FKBP8 for 48 hours. UCP2 degradation was measured as in Fig. 1. Proteins (1×105 cells/lane) were separated by SDS-PAGE and immunoblotted for UCP2 and β-actin. Values are means ± s.e.m. (n=4), corrected for loading (β-actin). All statistical significances were determined by repeated measures ANOVA (comparison of matching non-zero time points) with Dunnett's post-hoc testing (*P<0.05, **P<0.01, ***P<0.001). (B) INS-1E cells were treated with PIC-1 for 2 hours and fractionated using the Q Proteome cell compartment kit. Proteins were separated by SDS-PAGE and immunoblotted for UCP2. Values are normalised to the cell numbers used to generate the fractions and show means ± s.e.m. (n=3). Statistical significance was determined by Student's t-test. Significant P values are shown.

Article Snippet: Immunoblotting Typically, 1×10 5 cells or 25 μg mitochondrial protein in gel loading buffer (10% (w/v) SDS, 250 mM Tris-HCl (pH 6.8), 5 mM EDTA, 50% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 0.05% (w/v) Bromophenol Blue) were separated by 12.5% SDS-PAGE, transferred onto a Protran nitrocellulose membrane (Whatman) using the semi-dry method (20 V for 30 minutes), and probed with 0.2 μg/ml anti-human UCP2 goat polyclonal IgG antibody (Santa Cruz Biotechnology, cat. no. sc-6525), 0.2 μg/ml anti-human adenine nucleotide translocase (ANT) goat polyclonal IgG antibody (Santa Cruz Biotechnology, cat. no. sc-9300), 0.2 μg/ml anti-human β-actin rabbit polyclonal IgG antibody (Abcam, UK, cat. no. ab8227), or 0.2 μg/ml anti-HA (Abcam, UK, cat. no. ab9110).

Techniques: SDS Page, Comparison

Journal: Journal of Cell Science

Article Title: Degradation of an intramitochondrial protein by the cytosolic proteasome

doi: 10.1242/jcs.060004

Figure Lengend Snippet: Reconstitution of UCP2 degradation in vitro. Isolated INS-1E mitochondria (A,B) or mitoplasts (C) (240 μg per 260 μl) in sucrose-HEPES buffer (pH 7.4) were incubated at 37°C together with (as indicated) an ATP regeneration system (0.5 mM ATP, 10 mM phosphocreatine and 0.5 μg creatine kinase), ubiquitin mix (70 μg ubiquitin, 1.4 μg fraction 1, 1.4 μg fraction 2), 3.5 μg 26S proteasome fraction, 20 mM succinate, 50 μM PIC-1, and 20 μM FCCP. Aliquots were removed at the time points shown. Proteins (25 μg/lane) were separated by SDS-PAGE and immunoblotted for UCP2. Values are means ± s.e.m. (n=5), corrected for loading (Coomassie-Blue-stained membranes). All statistical significances were determined by repeated measures ANOVA (comparison of matching non-zero time points) with Dunnett's post-hoc testing (*P<0.05, **P<0.01, ***P<0.001). Typical UCP2 immunoblots (molecular mass ~30 kDa) are shown in supplementary material Fig. S4.

Article Snippet: Immunoblotting Typically, 1×10 5 cells or 25 μg mitochondrial protein in gel loading buffer (10% (w/v) SDS, 250 mM Tris-HCl (pH 6.8), 5 mM EDTA, 50% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 0.05% (w/v) Bromophenol Blue) were separated by 12.5% SDS-PAGE, transferred onto a Protran nitrocellulose membrane (Whatman) using the semi-dry method (20 V for 30 minutes), and probed with 0.2 μg/ml anti-human UCP2 goat polyclonal IgG antibody (Santa Cruz Biotechnology, cat. no. sc-6525), 0.2 μg/ml anti-human adenine nucleotide translocase (ANT) goat polyclonal IgG antibody (Santa Cruz Biotechnology, cat. no. sc-9300), 0.2 μg/ml anti-human β-actin rabbit polyclonal IgG antibody (Abcam, UK, cat. no. ab8227), or 0.2 μg/ml anti-HA (Abcam, UK, cat. no. ab9110).

Techniques: In Vitro, Isolation, Incubation, Ubiquitin Proteomics, SDS Page, Staining, Comparison, Western Blot

Journal: Journal of Cell Science

Article Title: Degradation of an intramitochondrial protein by the cytosolic proteasome

doi: 10.1242/jcs.060004

Figure Lengend Snippet: Model of UCP2 degradation via the ubiquitin-proteasome system. At the mitochondrial outer membrane (MOM), the proteasome − tethered by FKBP8 − recognises UCP2 that has been polyubiquitylated by an unidentified putative E3 ligase. The proteasome cap participates in the unfolding and extraction of UCP2 from the mitochondrial inner membrane (MIM) by processes that may be ATP- or ΔΨm-dependent. The cap also catalyses de-ubiquitylation, and the ubiquitin (Ub) is recycled. UCP2 is subsequently degraded by the peptidase activity of the proteasome core.

Article Snippet: Immunoblotting Typically, 1×10 5 cells or 25 μg mitochondrial protein in gel loading buffer (10% (w/v) SDS, 250 mM Tris-HCl (pH 6.8), 5 mM EDTA, 50% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 0.05% (w/v) Bromophenol Blue) were separated by 12.5% SDS-PAGE, transferred onto a Protran nitrocellulose membrane (Whatman) using the semi-dry method (20 V for 30 minutes), and probed with 0.2 μg/ml anti-human UCP2 goat polyclonal IgG antibody (Santa Cruz Biotechnology, cat. no. sc-6525), 0.2 μg/ml anti-human adenine nucleotide translocase (ANT) goat polyclonal IgG antibody (Santa Cruz Biotechnology, cat. no. sc-9300), 0.2 μg/ml anti-human β-actin rabbit polyclonal IgG antibody (Abcam, UK, cat. no. ab8227), or 0.2 μg/ml anti-HA (Abcam, UK, cat. no. ab9110).

Techniques: Ubiquitin Proteomics, Membrane, Extraction, Activity Assay