CD14 monocytes from 10 PBMC and 12 IHMC donors were isolated as described above (PBMC: n = 5 male, 5 female; IHMC: n = 4 male, 8 female). Cells were lysed with RIPA Buffer with Protease Inhibitors (Thermo Scientific) according to manufacturer description. Cell lysate was run on a 12% reducing acrylamide gel and transferred to a PVDF membrane. The membrane was blocked for 90 minutes with MilliQ water with 5% skim milk. TOLLIP (Biolegend) and actin (BD Biosciences) staining was done 1:1000 and 1:10,000, respectively, in TBST with 2% BSA for 1 hour. Secondary staining with HRP conjugated anti-murine IgG at 1:2000 (Biolegend) in TBST with 2% skim milk was done for 1 hour. TOLLIP and actin staining was measured using ECL detection reagents (Bio-Rad) and imaged with a ChemiDoc Imaging System (Bio-Rad).
Ripa buffer with protease inhibitor
Manufactured by Thermo Fisher Scientific
RIPA buffer with protease inhibitor is a lysis buffer used to extract proteins from cells and tissues. It contains a combination of detergents, salts, and protease inhibitors that help to solubilize and preserve proteins during the extraction process.
Automatically generated - may contain errors
Lab products found in correlation
Oligomycin, by Cayman Chemical (2 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Cayman Chemical (2 mentions)
Antimycin, by Cayman Chemical (2 mentions)
Seahorse xfp plates, by Agilent Technologies (2 mentions)
Seahorse xf mini instrument, by Agilent Technologies (2 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (2 mentions)
Actin, by Bio-Rad (1 mentions)
Ecl detection reagent, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Actin, by BD (1 mentions)
Enhanced chemiluminescence system, by GE Healthcare (1 mentions)
Anti mcp 1, by Cell Signaling Technology (1 mentions)
Anti tnf α, by Cell Signaling Technology (1 mentions)
Anti ph p38, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Anti il 6, by Cell Signaling Technology (1 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
Anti p38, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
6 protocols using ripa buffer with protease inhibitor
1
Quantification of TOLLIP Protein Expression
2
Molecular Signaling Pathways in HUVEC Activation
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After indicated treatments, RIPA buffer with protease inhibitors (Thermo Fisher Scientific) was added to HUVECs. Thereafter, equal amounts (about 80ug) of total protein samples were separated though 4-20% SDS-PAGE and transferred to PVDF membranes (Merck Millipore) using electroblotting. The membranes were blocked with 5% non-fat milk at the room temperature for 2 h and then incubated overnight at 4 °C with anti-Ph-p38 (1:1000, Cell Signaling Tech #4511), anti-p38 (1:1000, Cell Signaling Tech #9212), anti-Ph-NF-κB p65 (1:1000, Cell Signaling Tech #3033),anti-NF-κB p65 (1:1000, Cell Signaling Tech #8242),anti-VWF (1:1000, Cell Signaling Tech #65707), anti-IL-6 (1:1000, Cell Signaling Tech #12153), anti-TNF-α (1:1000, Cell Signaling Tech #6945),anti-MCP-1 (1:1000, Cell Signaling Tech #81599) and anti-GAPDH (1:1000, Cell Signaling Tech #5174) primary antibodies. The membranes were washed next day and then incubated with HRP-conjugated secondary antibodies (1:1000, Cell Signaling Tech #7074) for 2 h at the room temperature. Finally, an enhanced chemiluminescence system (GE Healthcare) was used to visualize the protein bands. The protein levels were presented as fold change relative to the expression of control. All Western assays were performed in duplicates or triplicates and all experiments were repeated at least three times.
3
Mitochondrial Function Profiling in Neurons
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Primary rat neurons or human i3Neurons were plated on Seahorse XFp plates (Agilent) at a density of 40,000 cells/well, then either treated with 2DG or transduced with codon-optimized lentiviruses as described in previous sections. 48 h after 2DG treatment or 4 days after transduction, the Seahorse Extracellular Flux assay was performed using the Seahorse XF Mini instrument (Agilent) according to the manufacturer’s instructions. During the assay, the cells were treated sequentially with each of the following mitochondrial toxins: oligomycin (1.5 μg/ml), FCCP (3 μM), and antimycin (1 μM) (all Cayman Chemical). Immediately after the assay, the cells were lysed with RIPA buffer with protease inhibitor (Thermo Scientific). The lysates were analyzed for total protein content using the Pierce BCA protein assay (Thermo Scientific). Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) values were then normalized to total protein.
4
Striatal CB1 receptor expression analysis
5
Overexpression of circMYC in MDA-MB-231 Cells
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After 1 day with overexpression of circMYC, the RIPA buffer with protease inhibitor (Thermo Scientific) from Boston BioProducts was used to harvest protein lysates from the transfected MBA-MD231 cells. The cell lysate was centrifuged at 13,000g for 5 min followed by the removal of precipitates. The supernatant was subjected to BCA protein assay to determine the protein concentration. For western blot analysis, 30 μg of protein was used.
The transfected cells were harvested, and the RNAs were purified at day 4 via the TRIzol™-chloroform extraction. For one well in a six-well plate, 500 μL of TRIzol™ was added into the well to lyse cells and stabilize RNAs, followed by addition of chloroform. The upper layer of the phenol-chloroform was used for column-based RNA purification (RNeasy from Qiagen). One microgram of total RNA was used for cDNA synthesis by iScript™ cDNA synthesis kit from Bio-Rad, which contains both oligo dT and random hexamers. The levels of circMYC were detected by the following divergent primers which detect only circularized MYC transcript using RT-qPCR: F 5′-CATCAGCACAACTACGCAGC-3′ and R 5′-TCCAGCAGAAGGTGATCCAG-3′. 18s rRNA was used as internal control: F 5′-GTAACCCGTTGAACCCCATT-3′ and R 5′-CCATCCAATCGGTAGTAGCG-3′. For the cell proliferation assay, WST-1 from Sigma Aldrich was used from days 1, 2, 3, and 4.
The transfected cells were harvested, and the RNAs were purified at day 4 via the TRIzol™-chloroform extraction. For one well in a six-well plate, 500 μL of TRIzol™ was added into the well to lyse cells and stabilize RNAs, followed by addition of chloroform. The upper layer of the phenol-chloroform was used for column-based RNA purification (RNeasy from Qiagen). One microgram of total RNA was used for cDNA synthesis by iScript™ cDNA synthesis kit from Bio-Rad, which contains both oligo dT and random hexamers. The levels of circMYC were detected by the following divergent primers which detect only circularized MYC transcript using RT-qPCR: F 5′-CATCAGCACAACTACGCAGC-3′ and R 5′-TCCAGCAGAAGGTGATCCAG-3′. 18s rRNA was used as internal control: F 5′-GTAACCCGTTGAACCCCATT-3′ and R 5′-CCATCCAATCGGTAGTAGCG-3′. For the cell proliferation assay, WST-1 from Sigma Aldrich was used from days 1, 2, 3, and 4.
6
Mitochondrial Function Assay in Neurons
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Primary rat neurons or human i3Neurons were plated on Seahorse XFp plates (Agilent) at a density of 40,000 cells/well, then either treated with 2DG or transduced with codon-optimized lentiviruses as described in previous sections. 48 hours after 2DG treatment or 4 days after transduction, the Seahorse Extracellular Flux assay was performed using the Seahorse XF Mini instrument (Agilent) according to the manufacturer’s instructions. During the assay, the cells were treated sequentially with each of the following mitochondrial toxins: oligomycin (1.5 μg/ml), FCCP (3 μM), and antimycin (1 μM) (all Cayman Chemical). Immediately after the assay, the cells were lysed with RIPA buffer with protease inhibitor (Thermo Scientific). The lysates were analyzed for total protein content using the Pierce BCA protein assay (Thermo Scientific). Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) values were then normalized to total protein.
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