In 48 well plates, murine macrophage J774.A1 or RAW264.7 cells were co-incubated with assorted pharmacological compounds, including CC, AICAR or berberine for 3 hrs. The media containing the compounds was then removed. For Western blots, the compound-treated cells were washed 3 times with PBS, pH7.4, and lysed with RIPA buffer (G Biosciences), supplemented with a SigmaFAST protease inhibitor tablet (Sigma Chemical, Inc.) and phosphatase inhibitor cocktail 2 and cocktail 3 (Sigma Chemical, Inc.), and then harvested. The protein extracts were boiled for 5 min in sample buffer (Thermo Scientific, Inc.) containing 200 μM DTT, separated by SDS-PAGE, transferred onto PVDF membrane and blotted with the indicated antibodies. The blots were detected using an enhanced chemiluminescence kit (Pierce, Inc.). Blots were stripped and re-probed for assessment of protein levels using GAPDH (Glyceraldehyde 3-phosphage dehydrogenase) as a loading control. Densitometry of the blots was performed using ImageJ (http://rsbweb.nih.gov/ij/ ). For CFU assays, the treated cells were washed with fresh drug-free medium 3 times, and then infected with C. neoformans as described above. To test the effects of the examined chemicals on the activity or expression of fungal virulence determinants, we measured urease activity and capsule as described (Feder et al., 2015 (link); Kwon-Chung et al., 1987 (link)).
Ripa buffer
Manufactured by G Biosciences
Sourced in United States
RIPA buffer is a type of cell lysis buffer commonly used in protein extraction and purification procedures. It is designed to disrupt cell membranes and solubilize cellular proteins while maintaining their native structure and function.
Automatically generated - may contain errors
Lab products found in correlation
Bis tris precast gel, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Blocking buffer, by LI COR (2 mentions)
Mouse anti β actin, by Thermo Fisher Scientific (2 mentions)
Pen strep ampho, by Sartorius (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Sigmafast protease inhibitor tablet, by Merck Group (1 mentions)
Sample buffer, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail 2 and cocktail 3, by Merck Group (1 mentions)
Pierce bca kit, by Thermo Fisher Scientific (1 mentions)
Proteinsimple fluorchem e imager, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Grp75, by Cell Signaling Technology (1 mentions)
Signalfire enhanced chemiluminescence ecl reagent, by Cell Signaling Technology (1 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (1 mentions)
Irdye 680rd goat anti mouse igg, by LI COR (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
15 protocols using ripa buffer
1
Macrophage Assays for Antifungal Compounds
2
Western Blot Analysis of Cellular Proteins
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Total protein was isolated from the cell cultures following transfection (72 h). Protein lysates were prepared by lysing the cells in ice-cold RIPA buffer (G-Biosciences) supplemented with protease and phosphatase inhibitors (MilliporeSigma), which were diluted 1:10 v/v as per the manufacturer’s recommendations. Cell debris was removed by centrifugation at 16,000 × g at 4°C, and protein concentrations were determined using a Pierce BCA kit (Thermo Fisher Scientific). A sample (20–35 mg) of the supernatant protein was mixed with LDS buffer and DTT, incubated at 70°C for 10 min and resolved on a 4%–12% Bis-Tris PAGE gradient gel before being transferred to a polyvinylidene fluoride (PVDF) membrane. Following transfer, the membrane was blocked in 5% skim milk for 1 h, washed, and incubated at 4°C overnight with a rabbit 1° monoclonal antibody against human GRP78, GRP94, GRP75, or β-actin (all purchased from Cell Signaling Technology) at a 1:1,000 dilution. The membrane was subsequently washed and incubated with an anti-rabbit horseradish peroxidase (HRP)-conjugated 2° Ab (Cell Signaling Technology) for 1 h at room temperature at a 1:2,000 dilution. The bands were visualized using a SignalFire enhanced chemiluminescence (ECL) reagent (Cell Signaling Technology) on a ProteinSimple FluorChem E imager.
3
Quantification of Regnase-1 and Roquin-1 in T Cells
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Western blotting was used to assess protein level quantification of Regnase-1 and Roquin-1 in T cells. After primary expansion and prior to cryopreservation, 8 × 106 T cells were pelleted and lysed in ice cold RIPA buffer (G Biosciences) supplemented with protease inhibitor (Sigma-Aldrich) for 30 min on ice then centrifuged at 14,000 rpm for 15 min at 4 °C. All lysates were prepared at a concentration of 1 × 106 cells per 10 µL volume of lysis buffer. Whole-cell lysates were boiled for 10 min and then resolved on a 12% Bis-Tris precast gel (Thermofisher). Proteins were transferred onto a polyvinylidene difluoride membrane (Millipore) and then incubated in blocking buffer (LI-COR) for 30 min at room temperature. Primary antibody incubation using rabbit anti-Regnase-1 (Proteintech) or rabbit anti-Roquin-1 (Bethyl Laboratories) and mouse anti-β-actin (Invitrogen) was performed overnight at 4 °C, followed by secondary antibody incubation using IRDye® 800CW goat anti-rabbit IgG (LI-COR) and IRDye® 680RD goat anti-mouse IgG (LI-COR) for 45 min at room temperature.
4
Western Blot Analysis of Prostaglandin Pathway
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Total protein was collected from cells using RIPA buffer (G-Biosciences, St. Louis, MO, USA) with the addition of Halt Protease and Phosphatase Inhibitor (Thermo Fisher Scientific). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked in 5% nonfat milk, and primary antibody incubations were performed overnight at 4°C. The following primary antibodies were used: COX-1 (Cell Signaling, Danvers, MA, USA), COX-2 (Cell Signaling), PTGIR (LifeSpan BioSciences, Seattle, WA, USA), PTGIS (Abnova, Taipei City, Taiwan), and EFTUD2 (Proteintech Group, Rosemont, IL, USA). Horseradish peroxidase–labeled secondary antibodies were detected with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Pittsburgh, PA, USA) on X-ray film (Bioland Scientific, Paramount, CA, USA) or with the Bio-Rad ChemiDoc MP Imaging System.
5
Epitope-Tagged INO80 Variant Expression
6
Western Blot Analysis of PD-L1 Expression
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Cell protein was harvested using RIPA buffer (G Biosciences, St. Louis, MO, USA) supplemented with Halt protease inhibitor (Thermo Fisher Scientific, Grand Island, NY, USA). Protein concentration was measured using the DC protein assay (Bio‐Rad, Hercules, CA, USA). Electrophoretic separation of protein (12–20 μg/well) was performed using 4–15% gradient polyacrylamide gels (Bio‐Rad). Separated protein was transferred onto PVDF membranes (Bio‐Rad), which were then blocked for 1 h at room temperature in Tris‐buffered saline containing 0.1% tween (TBS‐T) with 5% fat‐free milk (Bio‐Rad), followed by overnight incubation at 4°C with rabbit anti‐human PD‐L1 antibody (Cell Signaling Technologies, #13684T, Danvers, MA, USA) (1:1000 dilution) or mouse anti‐human β‐actin antibody (1:10 000 dilution) (Cell Signaling Technology, #3700 S) in 5% fat‐free milk with TBS‐T. Membranes were washed in TBS‐T and incubated for 20 min at room temperature with a 1:2000 dilution of horseradish peroxidase‐conjugated goat anti‐rabbit antibody (Promega, #W4011, Madison, WI, USA) or rabbit anti‐mouse antibody (Cell Signaling Technology, #7076 S) in 5% milk with TBS‐T. Protein signals were developed using the WesternBright ECL HRP substrate (Advansta, #K‐12045, San Jose, CA, USA) and measured using a Chemi‐Doc MP scanner (Bio‐Rad).
7
Western Blot Analysis of Phosphorylated HSL
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Whole-tissue protein lysate was extracted in RIPA buffer (G-Biosciences) containing Halt proteinase inhibitor (Thermo Fisher Scientific, 78430) and phosphatase inhibitor (Thermo Fisher Scientific, 78420). Protein lysate was determined using the bicinchoninic acid assay (Pierce). Protein lysates were denatured in Laemmli buffer (BioRad), resolved by 4–12% Mini-PROTEAN TGX SDS–PAGE (BioRad) and transferred to a polyvinylidene difluoride membrane. The membrane was incubated with primary antibodies diluted in EveryBlot blocking buffer (BioRad) overnight at 4 °C and then incubated with secondary antibody anti-rabbit HRP (Jackson Immuno Research, 711-036-152, 1:10,000) or anti-mouse HRP (Jackson Immuno Research, 715-036-150, 1:10,000) diluted in EveryBlot at room temperature. The results were visualized using SuperSignal West Pico PLUS Chemiluminescent Substrate (Invitrogen). The following antibodies were used for immunoblotting: anti-p-HSL(Ser660) (Cell Signaling, 45804, 1:1,000), anti-HSL (Cell Signaling, 4107, 1:1,000), anti-α-tubulin (Abcam, 7291, 1:10,000). Specifically, HSL was blotted after stripping the p-HSL membrane.
8
Epitope-Tagged INO80 Variant Expression
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The full length human INO80 transcript variant 1 (Accession: NM_017553.2) was used for 3X FLAG-tagging at C terminus using CRISPR-Cas9 system in HEK293 cells. The stable monoclonal population was verified by sequencing. HEK293 cells having INO80-3X FLAG were cultured in DMEM media (Corning #10013-CM) with 10% FBS (GIBCO # 26-140-095), 1X pen-strep-ampho (Biological Industries #03033-1B) in T25 flask and after 24 hr supplemented with 0.5μg/ml of puromycin for selection. Four million cells were subcultured in 150 mm plates (10 plates) until they reached full confluence by regularly changing the media every 2 days. At full confluence, the media was aspirated, and cells were washed twice with PBS. The cells were harvested in 0.3 HGN buffer containing the PI cocktail. The cell pellets were flash frozen and processed for pulldown using M2 affinity beads by slight modification of the protocol as described earlier (Shen, 2004 (link)). For siRNA experiments, HEK293 cells were transfected with siRNA against PRMTs using lipofectamine RNAiMAX Transfection Reagent (Thermo Scientific #13778030) at 60%–70% confluence. Cells were harvested after 24 hr in RIPA buffer (G Biosciences #786-490) to observe the knockdown effect using immunoblotting.
9
Western Blot Analysis of Fibrosis Markers
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Anti-alpha SMA. (A2547; Sigma-Aldrich), anti-collagen (#90395; Abcam, Cambridge, MA), anti-TGF-β (#66043; Abcam), anti-PDGFR-β (sc-432; Santa Cruz Biotechnology, Santa Cruz, CA), anti-GAPDH (sc-1694; Santa Cruz Biotechnology), anti-p-MEK1/2 (#2338; Cell Signaling Technology, Danvers, MA), anti-MEK1/2 (#8727; Cell Signaling Technology), anti-p-ERK1/2(p44/42) (#4370; Cell Signaling Technology), anti-ERK (#4695; Cell Signaling Technology), and p-CREB (#9198; Cell Signaling Technology) were used for western blotting. Cell or tissues were lysed with RIPA buffer (G-Biosciences, St. Louis, MO, USA) with a protease inhibitor cocktail followed by western blotting as previously described41 (link). The protein concentration of the lysates was quantified by using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA) Denatured proteins were loaded into SDS–polyacrylamide gel, resolved by electrophoresis, and transferred to a nitrocellulose membrane, blocked by incubation with 3% bovine serum albumin (Sigma) for 1 h at room temperature, and the membrane was then incubated with each primary antibody overnight. Incubation with the appropriate secondary antibodies conjugated to HRP was performed for 1 h. Protein bands were developed onto an X-ray film (Fujifilm) after incubation with enhanced chemiluminescence.
10
Western Blot Analysis of Cell Cycle Regulators
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The B16F10 cells were seeded in 12-well plates at a density of 2 × 105 cells/well. The cells were treated with drugs for 24 h after 16 h of incubation. The cells were subsequently lysed in RIPA buffer (G-Biosciences, MO, U.S.A.) containing protease inhibitors (Roche Applied Science, Mannheim, Germany). The total extracted protein content (25 μg) was separated by SDS/PAGE, and transferred on to PVDF membranes. The PVDF membranes were incubated with primary antibodies at a dilution of 1:500 or 1:1000 to detect p21 (catalog number: GTX27960, GeneTex Inc., U.S.A.), p27 (catalog number: GTX55090, GeneTex Inc., U.S.A.), and β-actin (catalog number: GTX109639, GeneTex Inc., U.S.A.). The protein band density was quantitated by ImageJ software (National Institutes of Health, U.S.A.). The fold change in protein expression was expressed as a ratio calculated by dividing the p21 or p27 protein band density by the β-actin band density and then normalized to the control group.
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