Prior to lysis, cells were washed thrice with chilled PBS. The resulting lysate was centrifuged at 12,000 rpm for 15 min. Protein concentration in the supernatant was determined using the BCA Protein Assay Kit. Subsequently, SDS-PAGE separation was performed and the separated proteins were transferred to activated PVDF membranes. Blots were blocked with 5% skimmed milk in TBS for 1 h at room temperature, followed by overnight incubation with antibodies specific for β-actin (1:3000), IRE1α (1:1000), PERK (1:1000), ATF6 (1:1000), GRP78(1:4000), GSDMD-N (1:1000), Pro-Caspase1 (1:1000), Cleaved Caspase1 (1:1000), NLRP3 (1:1000), ASC(1:1000), IL-1β (1:1000), Cleaved-IL-1β (1:1000) or IL-18 (1:1000). Blots were then rinsed 6 times with TBST (5 min/wash), probed for 1 h with HRP-labeled goat anti-rabbit IgG (1:3000) at room temperature and rinsed 4 times with TBST and 2 times with TBS (5 min/wash), then immunoreactive bands were visualized using an ECL kit from Abclonal (Wuhan, China). Protein bands were scanned with an Amersham Imager 6000 (GE Healthcare, Fairfield, CT, USA).
Amersham imager 6000
Manufactured by GE Healthcare
Sourced in United States
The Amersham Imager 6000 is a high-performance imaging system designed for a variety of life science applications. It features a large field of view, high sensitivity, and flexible imaging modes to accommodate different sample types and detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ecl kit, by ABclonal (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by Abcam (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
β actin, by Abcam (1 mentions)
Cocktail of protease inhibitors, by Beyotime (1 mentions)
β tubulin, by Solarbio (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Secondary antibodies tagged with horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
β catenin, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
4 protocols using amersham imager 6000
1
Western Blot Analysis of UPR and NLRP3 Proteins
2
Western Blot Analysis of DLC1 Protein
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Each cell line was lysed with RIPA buffer with 0.1 M dithiothreitol, protease, and phosphatase inhibitors (Thermo Scientific, Waltham, MA, USA). The total protein was quantified using a BCA protein assay (Thermo Scientific, Waltham, MA, USA). The protein (25 µg) from each sample was loaded into 10% SDS-PAGE gel, separated by electrophoresis, and transferred onto a PVDF membrane (Biorad, Hercules, CA, USA). The membrane was blocked with 5% skimmed milk and incubated with primary antibody against DLC1 (Santa Cruz, CA, USA) and β-actin (Abcam, Cambridge, MA, USA) at 1:2000 overnight at 4 °C. Incubation with secondary antibody HRP (Abcam, Cambridge, MA, USA) at 1:5000 was performed on the next day for an hour at room temperature and visualized by chemiluminescence (ECL, Abcam, USA) with a gel doc Amersham Imager 6000 (GE Life Sciences, Buckinghamshire, UK). Band intensity on the PVDF membranes was analyzed with Image J software and normalized to β-actin level.
3
Protein Extraction and Western Blot Analysis
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Proteins were extracted from cells by using RIPA Lysis Buffer (Beyotime, China) supplemented with a cocktail of protease inhibitors (Beyotime, China) after rinsing cells three times with cold PBS. The lysates were then subjected to centrifugation at 12,000×g at 4 °C for 5 min. Next, the isolated protein samples were mixed with sample buffer and boiled for 5 min at 100 °C. The total protein concentrations were determined using a bicinchoninic acid protein assay kit (Beyotime, Jiangsu, China). Equivalent amounts of protein samples were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, Sigma, USA). After blocking with 5% skimmed milk, the membranes were incubated with the following antibodies: MMP17 (1:1000, Zenbio), SH3GL2 (1:1000, Zenbio), and β-Tubulin (1:1000, Solarbio). The membranes were then incubated with appropriately HRP-conjugated secondary antibodies (1:5000) and extensively rinsied with TBST. Immunoreactive bands were detected using a chemiluminescence kit (ECL Substrate kit; Abclonal, China), and the protein bands were captured using the Amersham Imager 6000 (GE Healthcare).
4
Pituitary Tumor Proteome Analysis
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Whole-cell proteins were extracted from pituitary tumor tissues or cells using ice-cold radioimmunoprecipitation assay lysis buffer (50 mM Tris, pH 7.5; 250 mM NaCl); 10 mM ethylenediaminetetraacetic acid; 0.5% NP-40; 1 mg/mL leupeptin; 1 mM phenylmethylsulfonyl fluoride; and 4 mM NaF (Sigma-Aldrich) containing protease and phosphatase inhibitor cocktails (Roche, Mannheim, Germany). Membranes were incubated with primary antibodies against MITF (1:800, Abcam), WIF-1 (1:2000, Abcam), b-catenin (1:3000, Abcam), or GAPDH (1:10,000, Beijing Guan Xing Yu Sci & Tech Co., Ltd, Beijing, China) overnight at 4°C. Blots were incubated with secondary antibodies tagged with horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX). Blots were visualized by enhanced chemiluminescence and densitometry was performed with an Amersham Imager 6000 (GE Health Care, Piscataway, NJ). To further determine differences in expression of b-catenin in the nucleus and cytoplasm, nuclear and cytoplasmic proteins were extracted separately using Nuclear-Cytosol Extraction Kits (Applygen Technologies Inc., Beijing, China). TBP levels (1:2000, Abcam) were used as loading controls for nuclear proteins and GAPDH was used for cytoplasmic proteins.
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