AGS and MKN1 cells were seeded at a density of 3–5 × 105 cells/well in 6-well plates and allowed to grow overnight. The cells were treated with Dimethyl sulfoxide (DMSO) as a control or PU1-1 at 5 μM and 7.5 μM concentrations for 24 h. Following the treatment, the cells were washed once with 1x phosphate-buffered saline (PBS) and lysed in RIPA buffer obtained from iNtRON Biotechnology, supplemented with a cocktail of protease and phosphatase inhibitors from Thermo Scientific. The protein concentrations were determined using the Pierce BCA protein assay kit provided by Thermo Scientific. Equal amounts of protein were resolved on 8–12% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in TBST (Tris-buffered saline with Tween) and then incubated overnight at 4 °C with primary antibodies. After washing, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The blots were washed five times with TBS-T and developed using an Enhanced chemiluminescent (ECL) detection system obtained from EMD Millipore.
Of protease and phosphatase inhibitors
Manufactured by Thermo Fisher Scientific
Sourced in United States
Protease and phosphatase inhibitors are chemical compounds used in biological research to prevent the degradation of proteins and the dephosphorylation of phosphorylated molecules. These inhibitors help maintain the integrity and activity of target proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by iNtRON Biotechnology (1 mentions)
Rabbit anti cathepsin d, by Cell Signaling Technology (1 mentions)
Mouse anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by GeneTex (1 mentions)
Rabbit anti actin, by Merck Group (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Succinate, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
Succinate, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Ibright cl1500 imaging system, by Thermo Fisher Scientific (1 mentions)
4 protocols using of protease and phosphatase inhibitors
1
Protein Expression Analysis in AGS and MKN1 Cells
2
Protein Expression Analysis in Cell Lysates
3
Succinate Modulates Collagen and p38 MAPK in NHDFs
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NHDFs were cultured in 6‐well plates and then treated with succinate (5 mmol/L‐ Sigma) or untreated in low FBS DMEM and after 48 hours the RNA was extracted and qPCR was performed for collagen1A1 and 18S. Data were normalized to 18S and was from 4 independent cultures. Primers for collagen1A1 forward 5′‐CAA GAG GAA GGC CAA GTC GAG G‐3′, reverse 5′‐ CGT TGT CGC AGA CGC AGA T‐3’. All samples were run in triplicate and samples lacking RT served as negative controls.
In other experiments, cells were treated with succinate (5 mmol/L) or untreated and after 15 minutes stimulation cells were lysed in RIPA buffer containing a cocktail of protease and phosphatase inhibitors (Thermo Fisher). The lysate was subjected to PAGE, and Western blotting was performed using phosphorylated P38 MAPK thy180‐ty182 residues 1:600 dilution (Cell signaling technology) and was reprobed with beta‐actin 1:20,000 dilution (Abcam).
In other experiments, cells were treated with succinate (5 mmol/L) or untreated and after 15 minutes stimulation cells were lysed in RIPA buffer containing a cocktail of protease and phosphatase inhibitors (Thermo Fisher). The lysate was subjected to PAGE, and Western blotting was performed using phosphorylated P38 MAPK thy180‐ty182 residues 1:600 dilution (Cell signaling technology) and was reprobed with beta‐actin 1:20,000 dilution (Abcam).
4
Western Blot Analysis of Ovarian Proteins
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Total ovarian proteins were isolated using RIPA buffer (Sigma, St. Louis, MO, USA) containing a cocktail of protease and phosphatase inhibitors (Thermo Fisher, Rockford, IL, USA). The samples were loaded onto 12% SDS polyacrylamide gels (20 µg of protein per lane) and separated via gel electrophoresis. Western blot analyses were performed as previously described in our studies [26 (link)]. All antibodies used are listed in Table 2 . A chemiluminescent reaction was induced using luminol with H2O2 as a substrate, and the signal was visualized using the iBright CL1500 Imaging System (Thermo Fisher Scientific, Rockford, IL, USA). Densitometric analysis of the blot images was performed via the ImageJ software package. The optical density (OD) of the bands of target proteins was compared to the OD of either β-actin (for StAR and CYP11A1) or total proteins (for 3β-HSD) from the same lane. The mean value of the calculated relative ODs of the Control group was set as 100% (± SEM) for each target protein (from n ≥ 5 Western blot analyses). The relative ODs of the Onset, Peak, and End groups were then calculated as a change in the Control values and presented as the mean change (%) ± SEM.
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