Phos-tag acrylamide (NARD, Hyogo, Japan) at a concentration of 5 mM and supplemented with 10 mM MnCl2 was added to the SDS-PAGE gels. Prior to blotting, the gels were first soaked in a transfer buffer containing 8 mM EDTA for 15 min to remove MnCl2, and the duration of blotting was extended to 3 h [36 (link)]. The VDAC1 (Sigma, USA) antibody at a 1 : 1000 dilution was then added to determine the extent of VDAC1 phosphorylation via Phos-tag gel blotting.
Vdac1
Manufactured by Merck Group
Sourced in United States
VDAC1 is a voltage-dependent anion-selective channel protein that is involved in the regulation of mitochondrial membrane permeability. It serves as a pore in the outer mitochondrial membrane, allowing the passage of small molecules and ions between the cytosol and the mitochondrial intermembrane space.
Automatically generated - may contain errors
Lab products found in correlation
Las 4000 luminescent image analyzer, by Fujifilm (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Anti gfap, by Agilent Technologies (1 mentions)
Transblot semi dry blotting, by Bio-Rad (1 mentions)
Anti sucla2, by GeneTex (1 mentions)
Anti abcb10, by GeneTex (1 mentions)
Anti abcb7, by GeneTex (1 mentions)
Anti rabbit igg h l hrp conjugate, by Promega (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Anti cytochrome c, by BD (1 mentions)
Anti tim23, by BD (1 mentions)
Total protein extraction kit, by Beyotime (1 mentions)
Tim23, by Affinity Biosciences (1 mentions)
Ha tag, by Proteintech (1 mentions)
Bradford protein quantification kit, by Bio-Rad (1 mentions)
Calnexin, by Affinity Biosciences (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Proteintech (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
4 protocols using vdac1
1
Phos-tag Gel Blotting for VDAC1 Phosphorylation
2
Astrocyte and Organelle Dynamics under Glucose Conditions
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To assess astrocyte activation, mitochondrial biogenesis and lysosome biogenesis after sustained NG and HG culture, mixed glia were lysed in Cell Lysis Buffer (Cell Signaling Technology, Beverly, MA, USA; Cat. 9803). After electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes (Millipore; Cat. IPVH00010) and the membranes were incubated overnight at 4°C with primary anti-GFAP (Dako, Tokyo, Japan, Cat. Z0334, 1:1,000), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; GeneTex, Irvine, CA, USA, Cat. GTX100118, 1:10,000), anti-LAMP1 (Abcam; Cat. ab24170, 1:1000), or anti-voltage-dependent anion-selective channel 1 (VDAC1; Millipore; Cat. MABN504; 1:1000) antibodies. After incubation with goat anti-rabbit (Sigma; Cat. A0545) or goat anti-mouse (Sigma; Cat. A9917) secondary antibodies, the immune complexes were detected using a chemiluminescence kit (Millipore; Cat. WBLUF0500) and the band intensities were quantified using analyzed by FUJIFILM LAS4000 luminescent image analyzer (Fujifilm Life Science, USA).
3
Immunoblot Analysis of Mitochondrial Proteins
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For immunoblots, between 7.5 and 25.0 μg of total protein as assessed by BCA assays (Pierce, Rockford, IL) were loaded onto SDS-PAGE gels and transferred by Transblot semi-dry blotting (BioRad, Hercules, CA). Antibodies used included Anti-SUCLA2 (GeneTex, Irvine, CA) at a dilution of 1:2,500, Anti-ABCB7 (GeneTex) at a dilution of 1:500, Anti-ABCB10 (GeneTex) at a dilution of 1:1,000, Anti-PPOX (generated in house by H.A.D. at U.G.A.) at a dilution of 1:2,000, Anti-FECH (generated in house by H.A.D. at U.G.A.) at a dilution of 1:30,000–50,000, Anti-HSP-60 (Sigma) at a dilution 1:50,000, Anti-Cytochrome C (BD Biosciences, San Jose, CA) at a dilution of 1:5,000, VDAC-1 (Millipore, Billerica, MA) at a dilution of 1:2,000 and Anti-TIM-23 (BD Bioscience) at a dilution of 1:2,000. Secondary antibodies used were Anti-Rabbit IgG (H+L) HRP Conjugate (Promega, Madison, WI) and Anti-mouse IgG (H+L) HRP conjugate at dilutions of 1:30,000–60,000 with SuperSignal West Pico Chemiluminescent substrate (Pierce) and X-ray film or ChemiDoc imaging system (BioRad) for detection.
4
Quantification and Localization of Proteins in Mouse Skin
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The protein samples of mouse skin or epidermal keratinocyte extracts were obtained using a total protein extraction kit (Beyotime, CHN). And then, the protein concentrations were detected using the Bradford Protein Quantification Kit (500-0205, Bio-Rad Laboratories). The samples were gone through the SDS-PAGE system and transferred to PVDF membranes (Millipore, USA). The membranes were incubated with specific primary antibodies overnight at 4 °C. The primary antibodies utilized were as follows. MAP4 (1:1000, Affinity, USA), p-MAP4 (1:1000, GL Biochem, CHN), p-MAP4 (S760) (1:1000, GL Biochem, CHN), p-MAP4 (S737) (1:1000, GL Biochem, CHN), LC3 (1:1000, sigma, USA), p62 (1:1000, sigma, USA), Beclin1 (1:1000, CST, USA), Bcl-2 (1:1000, CST, USA), VDAC1 (1:1000, Millipore, USA), Tomm20 (1:1000, Affinity, USA), TIM23 (1:1000, Affinity, USA), GM130 (1:1000, Affinity, USA), Calnexin (1:1000, Affinity, USA), HA-tag (1:1000, proteintech, USA) and β-Actin (1:1000, proteintech, USA). Then, corresponding secondary antibodies were used and the results were visualized using the ChemiDoc XRS System (Bio-Rad Laboratories). Each treatment was repeated 5 times, and the sample size for each time was 3.
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