Samples were lysed in lysis buffer (50 mM Tris (pH 7.4), 150 mM KCl, 1 mM EDTA, 1% NP-40, 5 mM NAM, 1 mM sodium butyrate, protease and phosphatase inhibitors). Proteins were separated by SDS–PAGE and transferred onto nitrocellulose or polyvinylidene difluoride membranes. Blocking and antibody incubations were performed in 5% BSA or non-fat dry milk. PGC-1α (ab54481 1:1000), UCP1 (ab10983 1:1000 for animal tissues, 1:500 for primary culture), TBX1 (ab109313 1:1000) and VDAC1 (ab14734 1:1000) antibodies were from Abcam; anti-CREB (9197 1:1000), p-CREB Ser133 (9191 1:1000), ERK (4695 1:1000), p-ERK (4376 1:1000), DRP1 (4E11B11 1:1000), p-DRP1 Ser616 (3455 1:1000) and p-DRP1 Ser637 (4867S 1:1000) antibodies were from Cell Signalling; anti-MFF antibody (17090-1-AP 1:1000) was from Proteintech group; anti-PARP1 (sc-7150 1:1000), GAPDH (sc-47724 1:2000) and TOMM40 (sc-11414 1:1000) antibodies were from Santa Cruz Biotechnology. The MitoProfile Total OXPHOS Rodent WB Antibody Cocktail (MS604-300 1:500) for mitochondrial subunits was purchased from MitoSciences. Antibody detection reactions were developed by enhanced chemiluminescence (Advansta) and imaged using the c300 imaging system (Azure Biosystems). Quantification was done using ImageJ software.
C300 imaging system
Manufactured by Azure Biosystems
Sourced in United States
The C300 imaging system is a digital imaging platform designed for high-quality image capture and analysis. It features a sensitive camera and advanced optics to provide clear and detailed images of various samples. The C300 system is capable of performing a range of imaging applications, but a specific description of its intended use is not available.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay, by Bio-Rad (3 mentions)
Dc protein assay, by Bio-Rad (3 mentions)
Pvdf membrane, by Roche (2 mentions)
Anti gapdh 14c10, by Cell Signaling Technology (2 mentions)
Ubiquitin, by Enzo Life Sciences (2 mentions)
Oxphos cocktail, by Abcam (2 mentions)
Pink1, by Novus Biologicals (2 mentions)
Lonp1, by Merck Group (2 mentions)
Phopsho p62, by Cell Signaling Technology (2 mentions)
Mtco1, by Abcam (2 mentions)
Hsp60, by Enzo Life Sciences (2 mentions)
Lc3a b, by Cell Signaling Technology (2 mentions)
Cofilin, by Cell Signaling Technology (2 mentions)
Andphospho cofilin, by Cell Signaling Technology (2 mentions)
Phospho mlc2 ser19, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
P erk 4376, by Cell Signaling Technology (1 mentions)
Drp1 4e11b11, by Cell Signaling Technology (1 mentions)
27 protocols using c300 imaging system
1
Protein Expression Analysis Protocol
2
Western Blot Analysis of IL-1β
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Lysates collected from in vitro infected BMDMs at different time points as described above (In vitro infection scheme and collection) were subjected to SDS-PAGE and gels were electrophoretically transferred onto polyvinylidine difluoride membranes (PVDF). Protein expression was examined using the following primary antibodies: anti-β-Actin and anti-IL-1β (D6A8, D3H1Z; Cell signaling technologies) were used with anti-rabbit HRP secondary antibody (Jackson Immuno Research, 111-035-144). Membranes were incubated in SuperSignal West Femto Maximum Sensitivity Substrate (ThermoScientific, 34096) and bands were visualized using an Azure Biosystems C300 imaging system.
3
Extracellular Vesicle Protein Profiling
4
Quantifying KpB308-2 Outer Membrane Enrichment
5
Protein Extraction and Immunoblotting from C.elegans, C2C12 Cells, and Mouse Tissues
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C.elegans were lysed by sonication with TBS buffer containing protease and phosphatase inhibitors (Roche), and analyzed by western and dot blot. The concentration of extracted protein was determined using the Bio-Rad Protein Assay. Membranes were blocked in 10% milk for 2h or overnight. Homogeneous loading was monitored by ponceau red. Antibody detection reactions for all immunoblot experiments were developed by enhanced chemiluminescence (Advansta) and imaged using the c300 imaging system (Azure Biosystems). Each immunoblot experiment was repeated at least twice using at least three biological replicates each containing approximately 1,000 worms.
C2C12 cell lysates were prepared for immunoblotting as for C.elegans. Each experiment was repeated at least twice using 3 biological replicates.
For mouse tissues, frozen forelimbs samples were lysed by mechanical homogenization with RIPA buffer containing protease and phosphatase inhibitors for western blot analysis. Lysates were prepared and analyzed by dot blot as described for worms.
C2C12 cell lysates were prepared for immunoblotting as for C.elegans. Each experiment was repeated at least twice using 3 biological replicates.
For mouse tissues, frozen forelimbs samples were lysed by mechanical homogenization with RIPA buffer containing protease and phosphatase inhibitors for western blot analysis. Lysates were prepared and analyzed by dot blot as described for worms.
6
Western Blot Analysis of Muscular Dystrophy
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DMD patient myoblast cells or mouse skeletal muscle tissues were lysed on ice in radioimmunoprecipitation assay buffer composed of 50 mM tris-HCl, 5 M NaCl, 5 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), 100 mM NaF, sodium deoxycholate (5 mg/ml), and 1% NP-40 containing protease and phosphatase inhibitors (Roche). Protein concentrations were determined using the Bradford method, and samples were loaded on a 12% SDS–polyacrylamide gel electrophoresis (PAGE) gel. After electrophoresis, proteins were separated by SDS-PAGE and transferred onto methanol-activated polyvinylidene difluoride membranes. Blocking of the membranes was done in 5% milk-TBST (tris-buffered saline with 0.1% Tween 20) for 1 hour, and after washing, the membranes were incubated overnight with primary antibody anti-SPTLC1 (Proteintech) or anti-SPTLC2 (Thermo Fisher Scientific) or anti-CERS2 (Sigma-Aldrich) in 3% BSA-TBST (1:1000). Incubation with secondary anti-rabbit polyclonal antibody was done in 5% BSA-TBST (1:2000). Antibody detection reactions were developed by enhanced chemiluminescence (Advansta) and imaged using the c300 Imaging System (Azure Biosystems).
7
Western Blotting of Signaling Pathways
8
Mitochondrial Protein Analysis by Western Blot
9
Extracting β-Tubulin from N. bombycis
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109 mature spores of N. bombycis suspended in 400 μL of lysis buffer (Beyotime) were mixed with 0.4 g of glass beads (212–300 μm, acid-washed, Sigma-Aldrich) and disrupted by violent oscillation. After centrifugation at 12,000 g for 10 min, the supernatants were isolated and measured for immunoblotting. Proteins were separated on a 12% SDS-PAGE and transferred to PVDF membrane (Roche). The membranes were treated as follows: blocking for 1 h at 37°C in Blocking Buffer (Beyotime), washing three times, incubating with a 1:9,000 dilution of β-Tubulin antiserum for 1 h at 37°C, washing three times, incubating with 1:8,000 peroxidase-labelled goat anti-mouse IgG (Roche) for 1 h at 37°C, washing three times, and incubating with Pierce™ ECL Western Blotting Substrate for 1 min, followed by imaging using a Azure Biosystems C300 imaging system.
10
Inflammatory Cytokine Production in RAW 264.7 Cells
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C. acnes (4 × 108 cfu/mL) was heated to 80 °C for 30 min to kill the bacteria. HKC was freeze-dried as a powder and maintained at 4 °C until use. RAW 264.7 cells were cultured in DMEM with 10% FBS, 1% penicillin-streptomycin, and 1% l -glutamine at 37 °C in a 5% CO2 atmosphere. RAW 264.7 cells (4 × 105 cells/mL) were seeded in six-well plates and co-treated with HKC (300 μg/mL) and test samples. NO and PGE2 production were determined according to previously described methods [20 (link)]. Whole-cell lysates were prepared via lysis with radioimmunoprecipitation assay buffer. The proteins in the lysate (30 μg) were denatured in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and analyzed by 10% SDS-PAGE. Gels were transferred to nitrocellulose membranes and probed with primary iNOS (clone N-20), COX-2 (clone C-20), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; clone 6C5) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Membranes were visualized with a BCIP/NBT kit (Gibco BRL, Grand Island, NY, USA). The level of protein expression was quantified using an Azure Biosystem C300 Imaging System. GAPDH expression was used as an internal control to compare the protein expression of iNOS and COX-2.
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