The liver and ileal tissues were homogenized in radioimmunoprecipitation assay (Solarbio, cat. R0020, China) buffer with phenylmethanesulfonyl fluoride (Cell Signaling Technology, cat. 8553, USA). The concentration of total protein was determined using a bicinchoninic acid (BCA) assay kit (Tiangen, Beijing, China). Equal quantities of protein extracts were resolved via 6–2% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene fluoride membranes. The membranes were blocked in 5% non-fat milk and subsequently incubated overnight at 4°C with primary antibodies. Then, a secondary horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody (ZSGB-BIO, China) was applied, and images were developed via an enhanced chemiluminescence (ECL) detection kit (Thermo Fisher Scientific, USA).
Phenylmethanesulfonyl fluoride
Manufactured by Cell Signaling Technology
Sourced in United States
Phenylmethanesulfonyl fluoride (PMSF) is a serine protease inhibitor commonly used in laboratory settings. It functions by irreversibly inhibiting the activity of serine proteases, which are enzymes involved in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Cell lysis buffer, by Cell Signaling Technology (5 mentions)
Ecl detection kit, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay (ripa), by Solarbio (1 mentions)
Bca assay kit, by Tiangen Biotech (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Tgf β1, by Cell Signaling Technology (1 mentions)
Phosphor smad3, by Cell Signaling Technology (1 mentions)
Col1a1, by Cell Signaling Technology (1 mentions)
α sma, by Cell Signaling Technology (1 mentions)
Smad3, by Cell Signaling Technology (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated donkey anti rabbit igg, by GE Healthcare (1 mentions)
Sheep anti mouse igg, by GE Healthcare (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cytiva (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Myd88, by Abcam (1 mentions)
Perilipin, by Cell Signaling Technology (1 mentions)
8 protocols using phenylmethanesulfonyl fluoride
1
Western Blot Analysis of Liver and Ileal Proteins
2
Skin Protein Extraction and Western Blotting
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The total protein of the skin was prepared in Cell Lysis Buffer plus 1 mM phenylmethanesulfonyl fluoride (Cell Signaling Technology, Danvers, MA, USA). Tissues were homogenized with 2 metal beads at a speed of 60 Hz for 2 min. After centrifugation at 12,000 × g for 20 min at 4 °C, the protein concentration was measured by the Bradford method, western blotting was done as described previously [9 (link)]. The primary antibodies for TGF-β1, α-SMA, COL1A1, phosphor-SMAD3, SMAD3, phospho-ERK1/2 (Thr202/Tyr204) (p-ERK), ERK1/2 and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA).
3
Western Blot Protein Analysis Protocol
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Cells were lysed in cell lysis buffer (Cell Signaling Technology) with 1 mM Phenylmethanesulfonyl Fluoride (Cell Signaling Technology). The protein concentration of each cell lysate was estimated by BCA assay (Pierce Chemical). Cell lysates containing total 20 µg of protein were applied to 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Invitrogen, Grand Island, NY). After being blocked with Tris buffered saline with 0.2% Tween-20 (TBST) containing 5% BSA or skim milk for 4 hours at room temperature, the membranes were incubated with antibody in appropriate dilution at 4°C. Horseradish peroxidase–conjugated donkey anti–rabbit IgG or sheep anti-mouse IgG (GE Healthcare, Piscataway, NJ) were used as secondary antibodies and incubated with the membranes at a 1/3000 dilution for 1 hour. β-actin was used as a loading control marker for normalization of each lane.
4
Hippocampal IL-6 Quantification Post-Surgery
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Three days after surgery, rats were sacrificed under isoflurane anesthesia. The hippocampus was harvested and stored at −80°C for further analysis. The hippocampus from one side was homogenized in cell lysis buffer (Cell Signaling Technology), mixed with phenylmethanesulfonyl fluoride (Cell Signaling Technology) and protease inhibitor (Thermo Fisher Scientific). Protein concentration was assayed with Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Interleukin IL-6 was measured using a commercially available ELISA kit following the manufacturer’s instructions (R&D Systems).
5
Western Blot Analysis of Murine Adipose Tissue
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Murine adipose tissue homogenates were lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) containing 1 mM phenylmethanesulfonyl fluoride (Cell Signaling Technology). Equal amounts of protein were fractionated by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Whatman GmbH, Dassel, Germany). Membranes were blocked in 5% BSA (in TBS/0.1% Tween-20) followed by incubation with antibodies against leptin (abcam), myeloid differentiation primary response protein-88 (MyD88; abcam), p53 (Santa Cruz Biotechnology), perilipin (PLIN; Cell Signaling Technology), PIAS1 (Cell Signaling Technology), preadipocyte factor-1 (PREF1; Thermo Fisher Scientific), or suppressor of cytokine signaling-1 (SOCS1; Novus Biologicals, Centennial, CO, USA). Antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Hy Test Ltd., Turku, Finland) were used to show total protein loading. Protein bands were visualized using horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, Chalfont St. Giles, UK) and detected with enhanced chemiluminescence substrate (Pierce Manufacturing, Appleton, WI, USA).
6
Western Blot Analysis of GPR30 Protein
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Cells were incubated with vehicle or 4-OHT as described previously. After 24 hours, the cells were lysed using Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA) supplemented with 1 mM phenylmethanesulfonyl fluoride (Cell Signaling Technology) and 10 µL Halt protease/phosphatase inhibitor cocktail (Santa Cruz Biotechnology, Dallas, TX). Protein was added to Bolt 4%–12% Bis-Tris Gels and run with Bolt MES SDS running buffer (Invitrogen, Carlsbad, CA). Samples were transferred to nitrocellulose membranes using the iBlot system (Invitrogen). Western blots were probed with the primary antibody human anti-GRP30 (1:250; Abcam, Cambridge, MA), human anti-GAPDH (1:1000; Cell Signaling Technology), mouse anti-GPR30 (1:500; ThermoFisher), or anti β-actin (1:1000; Cell Signaling Technology) at 4℃ for 18 hours. IRDye 800CW or 680RD secondary antibodies (Li-Cor Biosciences, Lincoln, NE) were used at a 1:10,000 dilution at room temperature for 1 hour. Membranes were visualized on a Li-Cor Odyssey Imager (Li-Cor Biosciences).
7
Determining PD-L1 Levels in Keratinocytes
8
Quantification of Active RAC1 Levels
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The activation of RAC1 was assessed by Active RAC1 Detection Kit (#8815, Cell Signaling Technology) according to the manufacturer's instruction. Briefly, cells were washed with ice-cold phosphate-buffered saline and lysed with lysis buffer that was freshly added with 1 mM phenylmethanesulfonyl fluoride (#8553, Cell Signaling Technology) on ice for 5 min. After centrifugation at 16 000 × g for 15 min at 4 °C, the supernatant was mixed with GST-PAK1-PBD/glutathione resins and incubated at 4 °C for 1 h. Then the resin was washed three times with 1× cell lysis/ binding/wash buffer. The resin-bound GTP-RAC1 was eluted with 2× SDS sample buffer containing 200 mM dithiothreitol followed by western blot analysis with a mouse against human RAC1 mAb (#8631, 1:1000, Cell Signaling Technology). Total RAC1 in cell lysate was also determined by western blotting.
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