Protein expressions of FOXP3 and RoR-γt in mice kidney tissues were quantified using Western blot analysis. Mice kidney tissues were lyzed by NP-40 Lysis Solution (N8032; Solarbio) which added with protease inhibitor mixture (P6730; Solarbio) and PMSF (P0100; Solarbio) to obtain the total protein in tissues. Then, the concentration of total protein was evaluated using a BCA protein assay kit (PC0020; Solarbio). After the protein was denatured by mixing with loading buffer (P1040; Solarbio) and 100 °C heating for 5 min, the protein was separated by the SDS-PAGE gel (P1200, Solarbio) and transferred onto PVDF membrane (YA1701, Solarbio). Then, the PVDF membrane was incubated with western blocking buffer (SW3010; Solarbio) for 2 h followed by incubating FOXP3 antibody (ab215206; 1:1000, 47 kDa, Abcam, Cambridge, UK), RoR-γt antibody (ab113434; 1:2000, 55 kDa, Abcam), and GAPDH antibody (ab8245; 1:10,000, 36 kDa, Abcam) for 16 h at 4 °C. On the second day, membrane was further incubated with relative goat-anti rabbit IgG (ab6721; 1:20,000, Abcam), goat-anti mouse IgG (ab6789; 1:10,000, Abcam), or donkey-anti goat IgG (ab6885; 1:10,000, Abcam) antibody for 2 h. At last, after the membrane was added with ECL Luminescent Liquid (M41129, MERYER), the protein signaling on the membrane was examined by Image Lab 3.0 detector (Bio-Rad, Hercules, California, USA).
P6730
Manufactured by Solarbio
Sourced in China
The P6730 is a laboratory equipment designed for general-purpose applications. It has a compact and durable construction.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ab215206, by Abcam (1 mentions)
Ab113434, by Abcam (1 mentions)
Ab6885, by Abcam (1 mentions)
Ab6789, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
P0100, by Solarbio (1 mentions)
P1200, by Solarbio (1 mentions)
Ya1701, by Solarbio (1 mentions)
P1040, by Solarbio (1 mentions)
Ab8245, by Abcam (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Ibright 750, by Thermo Fisher Scientific (1 mentions)
Ab181603, by Abcam (1 mentions)
Ab181373, by Abcam (1 mentions)
Sc 2005, by Santa Cruz Biotechnology (1 mentions)
Sc 2357, by Santa Cruz Biotechnology (1 mentions)
R0020, by Solarbio (1 mentions)
Ab76315, by Abcam (1 mentions)
7 protocols using p6730
1
Quantification of FOXP3 and RoR-γt Protein Expressions in Mice Kidneys
2
Examining Keap1-Nrf2 Protein Interactions
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Different plasmids (Keap1-Flag, Keap1α-Flag, Keap1β-Flag, and Nrf2-V5) were transfected into HepG2 cells (4 × 105) that had been allowed to grow for 24 h on 6-well plates. After 8 h of transfection, the cells were recovered for 24 h in a fresh medium. The experimental cells were lysed in the RIPA buffer (C1053, PPLYGEN) containing both protease and phosphatase inhibitors (Solarbio, P6730). The total lysates were centrifuged, and the resulting supernatants were subjected to coimmunoprecipitation (CO-IP) with antibodies against V5 or Flag (Invitrogen), before being pulled down by the BeyoMag™ Protein A+G beads (Beyotime [38 (link)], P2108-1ML). The eluted proteins were separated by SDS–PAGE. The protein-blotted membranes were probed with V5 or Flag antibodies and horseradish peroxidase-conjugated secondary antibodies to IgG (ZSGB-BIO, ZB-2305, 0.1ML), prior to being visualised by enhanced chemiluminescence reagents (Thermo, iBright 750, Shanghai, China [39 (link)]).
3
Western Blot Analysis of Kidney Proteins
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Total proteins of frozen kidney tissues and harvested cells were extracted with radioimmunoprecipitation buffer (R0020, Solarbio) with a protease inhibitor cocktail (P6730, Solarbio). After quantification with a BCA protein assay kit (No. 23227, Thermo Fisher), protein (20 μg) was separated by 8% or 10% SDS-PAGE and then transferred to a PVDF membrane. The membranes were blocked in 5% non-fat milk at room temperature (RT) for 1 h and then incubated with primary antibodies overnight at 4°C. Primary antibodies used were shown below: anti-Klotho (ab181373, Abcam; sc-515942, Santa Cruz), anti-DNA methyltransferase (DNMT) 1 (#5032, CST), anti-DNMT3a (#3598, CST), anti-DNMT3b (#67259, CST), anti-p-STAT3 (ab76315, Abcam), anti-α-smooth muscle actin (α-SMA; #14968, CST), anti-E-cadherin (E-cad; #14472, CST), and anti-GAPDH (ab181603, Abcam). The next day, membranes were incubated with HRP-labeled secondary antibodies (sc-2357 & sc-2005, Santa Cruz) for 1 h at RT. Immunoblot signals were detected and then analyzed by Image J software with normalization to GAPDH.
4
Immunoprecipitation and Western Blotting
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Cells were lysed in Pierce IP Lysis Buffer (87788, Thermo) with a protease inhibitor mixture (P6730, Solarbio) and phosphatase inhibitor (P1260, Solarbio). Protein A/G Magnetic Beads (HY-K0202, MCE) were added to the mixture for 180 min at 4 °C and then immunoprecipitated with specific antibodies overnight at 4 °C. Then, the protein-antibody complexes were incubated with Protein A/G Magnetic Beads for another 2–4 h, the protein–magnetic beads complexes were harvested by The Magna GrIPTM Rack (#20-400, Millipore). Finally, the magnetic beads were eluted by boiling in 1× SDS-PAGE loading buffer before western blotting.
5
Isolation and Analysis of Nuclear Proteins
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Nuclear protein was extracted using a nucleus isolation kit according to manufacturer’s instructions (Solarbio, Beijing, China, R0050). Briefly, 30 mg liver was homogenized with a glass Dounce homogenizer in 500 μL lysis buffer, supplemented with protease inhibitor mixture (Solarbio, Beijing, China, P6730). Lysate was centrifugated at 700× g for 10 min at 4 °C. Protein concentration in supernatant solution was determined and boiled with protein loading buffer at 95 °C. The denatured protein sample containing cytosolic proteins was used to run western blot analysis. The pellet containing the nucleus was dissolved using lysis buffer and mixed with medium buffer and centrifuged at 700× g for 10 min at 4 °C. The supernatant was then discarded and the pellet resolved using lysis buffer. The protein concentration was determined and the nuclear sample denatured with protein loading buffer at 95 °C.
6
Enzyme Activity Assays for PK and PTA
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Sample preparation for the enzyme activity assays of PK and PTA were performed using a modified version of the method described by Bergman et al. (2016) [21 (link)]. The strains were cultivated in a citrate fermentation medium. After being washed twice with water, the mycelia were immediately ground into a powder using liquid nitrogen. For each sample, 0.2 g of powder was collected and mixed with 1 mL of a protein extraction buffer (pH 7.0) that contained 50 mM histidine-HCl, 20 mM KH2PO4-Na2HPO4, 2 mM dithiothreitol, 1 mM MgSO4 and 1× Protease inhibitor mixture (Solarbio, P6730). The mixture was thoroughly blended and then centrifuged at 10,000× g for 10 min at 4 °C. The supernatant was stored on ice. The total protein concentrations were determined using the BCA Protein Assay Kit (TaKaRa, T9300A). The activities of PK and PTA were measured in 96-well microtiter plates using the same ferric hydroxamate assay methods described by Bergman et al. (2016) [21 (link)]. The substrate for the assays of PK activities was fructose-6-phosphate.
7
PASMC Proliferation Assay for PAH
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PASMC in good growth condition were taken, washed three times with sterile PBS, and the cells were incubated in a culture incubator (P6730, Solarbio) for 2 min after the addition of appropriate amount of trypsin, PASMC were taken out, and the digestion was terminated by the addition of DMEM complete medium, centrifugation was carried out at 1200 rpm for 3 min, the cells were resuspended by adding the appropriate amount of medium, and the cells were cultured at 3000 cells per well. Cells were added to 96-well plates, incubated overnight, and different drugs were added to pretreat the cells for 24 h. Pulmonary arterial hypertension was modeled as previously described, and 10 μL of CCK8 (CA1210, Solarbio) solution was added to each well after 24 h of incubation in an incubator, and the absorbance at 450 nm was measured by an enzyme marker (Flash, ReadMax 1200) after 3 h of incubation.
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