Journal: International Journal of Molecular Sciences

Article Title: Integrated Multilayer Omics Reveals the Underlying Mechanisms in Xylazine-Related Heart Injury in Rats

doi: 10.3390/ijms26178532

Figure Lengend Snippet: Validation of related proteins in the fructose and mannose metabolism signaling pathway and cholesterol metabolism signaling pathway. ( A ) Western blot image of VDAC3. ( B ) Quantification of VDAC3. ( C ) Western blot image of PFKM. ( D ) Quantification of PFKM. * p < 0.05 compared with the control group.

Article Snippet: The membranes were incubated in 5% skimmed milk for 2 h, and then in diluted primary antibodies against PFKM (1:2000, 55028-1-AP, Proteintech, Rosemont, IL, USA); VDAC3(1:500, 55260-1-AP, Proteintech), GAPDH (1:10,000, 60004-1-Ig, Proteintech), tubulin (1:10,000, GB15140-100, Servicebio) at 4 °C 12 h. They were incubated in Goat Anti-Rabbit IgG H&L/HRP (1:10,000, GB23303, Servicebio) or Goat Anti-Mouse lgG H&L/HRP (1:10,000, GB23301, Servicebio) for 1 h. A chemiluminescence instrument was used to image the bands and Image J v1.8.0.345 was used to calculate the gray values.

Techniques: Biomarker Discovery, Western Blot, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Hypothermia-Induced Ubiquitination of Voltage-Dependent Anion Channel 3 Protects BV2 Microglia Cells From Cytotoxicity Following Oxygen-Glucose Deprivation/Recovery

doi: 10.3389/fnmol.2020.00100

Figure Lengend Snippet: Effect of hypothermia on the expression of apoptosis-associated proteins in microglial cells. (A) Representative western blot images showing the expression of caspase3, cleaved caspase3, and voltage-dependent anion channel 3 (VDAC3) in different groups. β-Actin was used as an internal control. (B) Bar graph showing the quantification of capase3/β-actin in different groups. (C) Bar graph showing the quantification of cleaved capase3/β-actin in different groups. (D) Bar graph showing the quantification of cleaved VDAC3/β-actin in different groups. Values show the mean ± SD ( n = 5). * P < 0.05, vs. Sham group; θ P < 0.05, vs. OGD group; φ P < 0.05, vs. OGD/R2h-HT groups; # P < 0.05, vs. OGD/R2h-NT groups; ▵ P < 0.05, vs. OGD/R4h-NT groups; ▴ P < 0.05, vs. OGD/R8h-NT groups.

Article Snippet: The materials used in this study are as follows: BV2 microglial cell lines (Procell, Wuhan, China); high glucose Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA); glucose-free DMEM (Jinuo Technologies, Hangzhou, China); 10% fetal bovine serum (FBS; Gibco Grand Island, NY, USA); cell counting kit-8 (CCK-8, Dojindo Molecular Technologies, Tokyo, Japan); 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1, Beyontime, Shanghai, China); tumor necrosis factor (TNF) α, interleukin (IL)-1β and (IL)-10 enzyme-linked immunosorbent assay (ELISA) kits (R & D Systems, Minneapolis, MN, USA); rabbit anti-induced nitric oxide synthase (iNOS) antibody (Proteintech, Wuhan, China); rabbit anti-arginase 1 (Arg1) antibody (Proteintech, Wuhan, China); mouse anti-ubiquitin antibody (Thermo Fisher Scientific, Waltham, MA, USA); rabbit anti-VDAC3 antibody (Aviva Systems Biology, San Diego, CA, USA); rabbit anti-caspase3 antibody (Proteintech, Wuhan, China); mouse anti-beta actin antibody (Abcam, Cambridge, UK); CoraLite594-conjugated goat anti-rabbit (red) IgG (Proteintech, Wuhan, China); CoraLite488-conjugated goat anti-mouse (green) IgG (Proteintech, Wuhan, China); horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Abcam, Cambridge, UK); SureBeads Protein G Magnetic Beads (Bio-Rad Laboratories, Hercules, CA, USA); RNAprep Pure Cell kit (Tiangen Biotech, Beijing, China); first strand cDNA synthesis kit (Abm, Richmond, BC, Canada); EvaGreen 2× quantitative polymerase chain reaction (qPCR) master mix kit (Abm, Richmond, BC, Canada).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Hypothermia-Induced Ubiquitination of Voltage-Dependent Anion Channel 3 Protects BV2 Microglia Cells From Cytotoxicity Following Oxygen-Glucose Deprivation/Recovery

doi: 10.3389/fnmol.2020.00100

Figure Lengend Snippet: Effects of hypothermia on the immunoreactivity of ubiquitin-positive and anti-VDAC3-positive microglia using double immunofluorescence assays. (A) Immunofluorescence images showing the BV2 microglia following OGD/R labeled with ubiquitin (green) and VDAC3 (red) antibody or secondary antibodies (control negative). Blue fluorescence indicates DAPI-labeled nuclei. Scale bar: 20 μm. (B) Quantification of the colocalization coefficient between ubiquitin and VDAC3. Values show the mean ± SD, n = 5. * P < 0.05, vs. sham group; θ P < 0.05, vs. OGD group; φ P < 0.05, vs. OGD/R2h-HT groups; # P < 0.05, vs. OGD/R2h-NT groups; ▴ P < 0.05, vs. OGD/R8h-NT groups.

Article Snippet: The materials used in this study are as follows: BV2 microglial cell lines (Procell, Wuhan, China); high glucose Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA); glucose-free DMEM (Jinuo Technologies, Hangzhou, China); 10% fetal bovine serum (FBS; Gibco Grand Island, NY, USA); cell counting kit-8 (CCK-8, Dojindo Molecular Technologies, Tokyo, Japan); 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1, Beyontime, Shanghai, China); tumor necrosis factor (TNF) α, interleukin (IL)-1β and (IL)-10 enzyme-linked immunosorbent assay (ELISA) kits (R & D Systems, Minneapolis, MN, USA); rabbit anti-induced nitric oxide synthase (iNOS) antibody (Proteintech, Wuhan, China); rabbit anti-arginase 1 (Arg1) antibody (Proteintech, Wuhan, China); mouse anti-ubiquitin antibody (Thermo Fisher Scientific, Waltham, MA, USA); rabbit anti-VDAC3 antibody (Aviva Systems Biology, San Diego, CA, USA); rabbit anti-caspase3 antibody (Proteintech, Wuhan, China); mouse anti-beta actin antibody (Abcam, Cambridge, UK); CoraLite594-conjugated goat anti-rabbit (red) IgG (Proteintech, Wuhan, China); CoraLite488-conjugated goat anti-mouse (green) IgG (Proteintech, Wuhan, China); horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Abcam, Cambridge, UK); SureBeads Protein G Magnetic Beads (Bio-Rad Laboratories, Hercules, CA, USA); RNAprep Pure Cell kit (Tiangen Biotech, Beijing, China); first strand cDNA synthesis kit (Abm, Richmond, BC, Canada); EvaGreen 2× quantitative polymerase chain reaction (qPCR) master mix kit (Abm, Richmond, BC, Canada).

Techniques: Ubiquitin Proteomics, Immunofluorescence, Labeling, Control, Fluorescence

Journal: Frontiers in Molecular Neuroscience

Article Title: Hypothermia-Induced Ubiquitination of Voltage-Dependent Anion Channel 3 Protects BV2 Microglia Cells From Cytotoxicity Following Oxygen-Glucose Deprivation/Recovery

doi: 10.3389/fnmol.2020.00100

Figure Lengend Snippet: Hypothermia induced ubiquitination of VDAC3 in microglial cells. (A) To detect non-specific binding with magnetic beads, OGD/R8h-HT cells were immunoprecipitated with IgG or anti-ubiquitin, followed by western blotting (WB) with anti-VDAC3 and anti-ubiquitin antibodies. Anti-ubiquitin and anti-VDAC3 were used for WB to determine the ubiquitination level of VDAC3 (left). WB data of ubiquitin, VDAC3, and β-actin from the input (right). (B) Immunoblot analyses of ubiquitinated VDAC3 in different groups. Cell lysates were immunoprecipitated with anti-ubiquitin antibody, followed by WB with the anti-ubiquitin antibody and VDAC3 antibodies. β-Actin was used as an internal control of input. (C) Quantification of ubiquitinated VDAC3 in experimental groups/sham group (Left) and ubiquitinated proteins/β-actin in different groups (right). Results are shown as mean ± SD ( n = 5). Left: * P < 0.05, vs. Sham group; θ P < 0.05, vs. OGD group; φ P < 0.05, vs. OGD/R2h-HT groups; # P < 0.05, vs. OGD/R2h-NT groups; ▴ P < 0.05, vs. OGD/R8h-NT groups.

Article Snippet: The materials used in this study are as follows: BV2 microglial cell lines (Procell, Wuhan, China); high glucose Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA); glucose-free DMEM (Jinuo Technologies, Hangzhou, China); 10% fetal bovine serum (FBS; Gibco Grand Island, NY, USA); cell counting kit-8 (CCK-8, Dojindo Molecular Technologies, Tokyo, Japan); 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1, Beyontime, Shanghai, China); tumor necrosis factor (TNF) α, interleukin (IL)-1β and (IL)-10 enzyme-linked immunosorbent assay (ELISA) kits (R & D Systems, Minneapolis, MN, USA); rabbit anti-induced nitric oxide synthase (iNOS) antibody (Proteintech, Wuhan, China); rabbit anti-arginase 1 (Arg1) antibody (Proteintech, Wuhan, China); mouse anti-ubiquitin antibody (Thermo Fisher Scientific, Waltham, MA, USA); rabbit anti-VDAC3 antibody (Aviva Systems Biology, San Diego, CA, USA); rabbit anti-caspase3 antibody (Proteintech, Wuhan, China); mouse anti-beta actin antibody (Abcam, Cambridge, UK); CoraLite594-conjugated goat anti-rabbit (red) IgG (Proteintech, Wuhan, China); CoraLite488-conjugated goat anti-mouse (green) IgG (Proteintech, Wuhan, China); horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Abcam, Cambridge, UK); SureBeads Protein G Magnetic Beads (Bio-Rad Laboratories, Hercules, CA, USA); RNAprep Pure Cell kit (Tiangen Biotech, Beijing, China); first strand cDNA synthesis kit (Abm, Richmond, BC, Canada); EvaGreen 2× quantitative polymerase chain reaction (qPCR) master mix kit (Abm, Richmond, BC, Canada).

Techniques: Ubiquitin Proteomics, Binding Assay, Magnetic Beads, Immunoprecipitation, Western Blot, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Hypothermia-Induced Ubiquitination of Voltage-Dependent Anion Channel 3 Protects BV2 Microglia Cells From Cytotoxicity Following Oxygen-Glucose Deprivation/Recovery

doi: 10.3389/fnmol.2020.00100

Figure Lengend Snippet: Effect of hypothermia on VDAC3 mRNA expression in microglial cells. The normalized VDAC3 mRNA level was determined in each group using real-time RT-PCR. * P < 0.05, vs. sham group. n = 5 repeated from five independent experiments.

Article Snippet: The materials used in this study are as follows: BV2 microglial cell lines (Procell, Wuhan, China); high glucose Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA); glucose-free DMEM (Jinuo Technologies, Hangzhou, China); 10% fetal bovine serum (FBS; Gibco Grand Island, NY, USA); cell counting kit-8 (CCK-8, Dojindo Molecular Technologies, Tokyo, Japan); 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1, Beyontime, Shanghai, China); tumor necrosis factor (TNF) α, interleukin (IL)-1β and (IL)-10 enzyme-linked immunosorbent assay (ELISA) kits (R & D Systems, Minneapolis, MN, USA); rabbit anti-induced nitric oxide synthase (iNOS) antibody (Proteintech, Wuhan, China); rabbit anti-arginase 1 (Arg1) antibody (Proteintech, Wuhan, China); mouse anti-ubiquitin antibody (Thermo Fisher Scientific, Waltham, MA, USA); rabbit anti-VDAC3 antibody (Aviva Systems Biology, San Diego, CA, USA); rabbit anti-caspase3 antibody (Proteintech, Wuhan, China); mouse anti-beta actin antibody (Abcam, Cambridge, UK); CoraLite594-conjugated goat anti-rabbit (red) IgG (Proteintech, Wuhan, China); CoraLite488-conjugated goat anti-mouse (green) IgG (Proteintech, Wuhan, China); horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Abcam, Cambridge, UK); SureBeads Protein G Magnetic Beads (Bio-Rad Laboratories, Hercules, CA, USA); RNAprep Pure Cell kit (Tiangen Biotech, Beijing, China); first strand cDNA synthesis kit (Abm, Richmond, BC, Canada); EvaGreen 2× quantitative polymerase chain reaction (qPCR) master mix kit (Abm, Richmond, BC, Canada).

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Neurology

Article Title: Effect of electroacupuncture on hippocampal protein lactylation in a rat model of vascular dementia

doi: 10.3389/fneur.2025.1629474

Figure Lengend Snippet: KEGG pathway clustering analysis of different comparison groups, the PPI network of differentially lactylated proteins in the pathways of interest, and verification of Vdac3 protein lactylation modifications. (A) Heatmap of lactylation sites for 12 key proteins. Each row represents a differentially modification site; each column represents a sample. Red indicates a high-level modification, blue indicates a low-level modification, and grey indicates sites that cannot be quantified in the corresponding samples. (B) The horizontal axis depicts different comparison groups, whereas the vertical axis indicates the enriched KEGG pathways. The colored blocks represent the functional descriptions of differentially expressed modified proteins enriched in each comparison group. Blue signifies high enrichment significance, whereas blue-white signifies low enrichment significance. * p < 0.05, ** p < 0.01, and *** p < 0.001. (C) PPI network of differentially lactylated proteins involved in the 4-VO group and sham group in the five pathways of interest. (D) PPI network in the 4-VO group and 4-VO + EA group. The color of the outer circle indicates the corresponding KEGG pathway of the protein, whereas the shape within the circle denotes the upregulation or downregulation of the modification sites contained within the protein. A diamond shape signifies proteins with upregulated modification sites, a downward arrow indicates proteins with downregulated modification sites, and a circle signifies proteins with upregulated and downregulated modification sites. The red box indicates the Vdac3 protein. (E) The IP experiment verified the lactylation modification level of Vdac3. (F) Quantification was performed using ImageJ software, and the values are expressed as the mean with the corresponding standard error from three replicate experiments ( n = 3, compared with the sham group, ## p < 0.01; compared with the 4-VO group, ** p < 0.01). 4-VO, four-vessel occlusion; EA, electroacupuncture; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein–protein interaction; IP, immunoprecipitation; Vdac3, mitochondrial voltage-dependent anion channel protein3.

Article Snippet: One milliliter of total protein extract was pre-cleared with 40 μL of protein A/G agarose beads by rotation at 4 °C for 1 h. Subsequently, the supernatant was incubated with 5 μL of Vdac3 antibody (A04802-2, BosterBio, China) overnight at 4 °C with rotation.

Techniques: Comparison, Modification, Functional Assay, Software, Immunoprecipitation

Journal: Frontiers in Neurology

Article Title: Effect of electroacupuncture on hippocampal protein lactylation in a rat model of vascular dementia

doi: 10.3389/fneur.2025.1629474

Figure Lengend Snippet: Analysis of lactylation site motifs in different groups and representative mass spectra of key proteins. (A) A heatmap of amino acids adjacent to lysine lactylation sites, with a green/red scale (−5 to 5) indicating the frequency of amino acid detection; red indicates high frequency; green indicates low frequency. The height ratio of amino acid abbreviation letters at specific positions reflects motif characteristics, with a % difference greater than 0 indicating a higher frequency of that amino acid at that position compared to the background, and less than 0 indicating a lower frequency. (B) Four conserved motif sites of lysine. (C) Sequence motif map of lactylation modification, 10 amino acids upstream and downstream of the modification site were selected, and the vertical axis indicates the difference between the proportion of the various amino acids at the same site in the experimental data set and the reference background, that is, percentage difference (%difference). The height ratio of amino acid abbreviation letters at specific positions represents the motif characteristics, and a %difference greater than 0 indicates that the frequency of the amino acid at this position is higher than the background. Values lower than 0 indicate that the frequency of the amino acid at this position is lower than the background. (D–F) The lactylation site of Vdac3 was identified by mass spectrometry. PPI, protein–protein interaction; 4-VO, four-vessel occlusion; EA, electroacupuncture.

Article Snippet: One milliliter of total protein extract was pre-cleared with 40 μL of protein A/G agarose beads by rotation at 4 °C for 1 h. Subsequently, the supernatant was incubated with 5 μL of Vdac3 antibody (A04802-2, BosterBio, China) overnight at 4 °C with rotation.

Techniques: Sequencing, Modification, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Voltage-dependent Anion Channels Modulate Mitochondrial Metabolism in Cancer Cells

doi: 10.1074/jbc.M112.433847

Figure Lengend Snippet: VDAC knockdown decreases mitochondrial membrane potential in HepG2 cells. In A, relative abundance of mRNA for VDAC1, VDAC2 and VDAC3 in native HepG2 cells was determined by qPCR. In B, cells were transfected with non-target siRNA and siRNA against VDAC1, VDAC2 and VDAC3. After 48 h, mRNA expression of the corresponding VDAC isoform and protein expression of all three isoforms were assessed by qPCR and immunoblotting. In C at 48 h after transfection with non-target siRNA and siRNA against VDAC1, VDAC2, and VDAC3, HepG2 cells were loaded with TMRM and imaged. Note decrease of mitochondrial TMRM fluorescence after knockdown of each VDAC isoform, but most markedly after VDAC3 knockdown. Image intensity was pseudocolored according to the reference bar. Arrows identify 4-μm fiduciary fluorescent beads. In D, average TMRM fluorescence after knockdown was plotted in comparison to transfection with non-target siRNA. *, p < 0.05 from five independent experiments analyzing 4–5 random fields containing 5–10 cells.

Article Snippet: Blots were blocked for 60 min in 5% albumin and probed with antibodies against VDAC1 (SC-8828, Santa Cruz Biotechnologies, Santa Cruz, CA, 1:200); VDAC2 (Ab-47104, Abcam, Cambridge, MA, 1:500); VDAC3 (MSA03/E0836, MitoSciences, Eugene, OR, 1:1000); and β-actin (691002, MP Biomedicals, Solon, OH, 1:2000).

Techniques: Knockdown, Membrane, Transfection, Expressing, Western Blot, Fluorescence, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Voltage-dependent Anion Channels Modulate Mitochondrial Metabolism in Cancer Cells

doi: 10.1074/jbc.M112.433847

Figure Lengend Snippet: VDAC3 knockdown decreases ATP, phosphorylation potential, adenine nucleotides, and NAD(P)H redox state. In A--C, adenine nucleotides and phosphate in HepG2 cells were measured after siRNA knockdown of VDAC1, VDAC2, and VDAC3, as described under “Experimental Procedures.” In D, NAD(P)H autofluorescence was imaged by multiphoton microcopy. Note that VDAC3 but not VDAC1 or VDAC2 knockdown decreased mitochondrial NADH autofluorescence. In E, average NAD(P)H autofluorescence after knockdown of VDAC isoforms is plotted in comparison to transfection with non-target siRNA. In F, NAD(P)H autofluorescence before and after addition of CCCP plus oligomycin or myxothiazol was measured for HepG2 cells transfected with non-target siRNA and siRNA against VDAC3. *, p < 0.05 compared with Non-target or Baseline from three independent experiments analyzing 4–5 random fields containing 5–10 cells.

Article Snippet: Blots were blocked for 60 min in 5% albumin and probed with antibodies against VDAC1 (SC-8828, Santa Cruz Biotechnologies, Santa Cruz, CA, 1:200); VDAC2 (Ab-47104, Abcam, Cambridge, MA, 1:500); VDAC3 (MSA03/E0836, MitoSciences, Eugene, OR, 1:1000); and β-actin (691002, MP Biomedicals, Solon, OH, 1:2000).

Techniques: Knockdown, Phospho-proteomics, Comparison, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Voltage-dependent Anion Channels Modulate Mitochondrial Metabolism in Cancer Cells

doi: 10.1074/jbc.M112.433847

Figure Lengend Snippet: VDAC 1, 2, and 3 isoforms have different sensitivity to blockade by tubulin. Double knockdowns of VDAC isoforms were performed in all possible combinations in HepG2 cells, and the remaining VDAC isoform was isolated and inserted into lipid bilayers. In A, normalized average conductance, G/Gmax, is plotted versus applied voltage for the three VDAC isoforms in the absence of tubulin. In B, tubulin (10 nm) was added to both sides of the membrane, which increased VDAC voltage-induced closure and decreased minimum conductance at > ± 40 mV for VDAC1 and 2 but did not affect VDAC3. In C, normalized conductance at −50 mV was plotted for all VDAC isoforms at tubulin concentrations of 10 nm and 50 nm, as indicated. *, p < 0.001 compared with no tubulin from 3–6 experiments for each group.

Article Snippet: Blots were blocked for 60 min in 5% albumin and probed with antibodies against VDAC1 (SC-8828, Santa Cruz Biotechnologies, Santa Cruz, CA, 1:200); VDAC2 (Ab-47104, Abcam, Cambridge, MA, 1:500); VDAC3 (MSA03/E0836, MitoSciences, Eugene, OR, 1:1000); and β-actin (691002, MP Biomedicals, Solon, OH, 1:2000).

Techniques: Isolation, Membrane