Anti-adiponectin receptor 1 (Abcam), anti–p-AMPK, anti-AKT (Cell Signaling), anti-β-actin, anti-AMPK, anti-caspase 3, anti-p-AKT, anti-Tau, anti-p-tau, anti-Aβ, anti-GFAP, anti-TNF-α, FITC-labelled goat-anti rabbit (Santa Cruz), anti-NeuN (Millipore), anti-iba-1 (Wako), insulin (Sigma), branched-PEI (Sigma), D-(+)- glucose solution (Sigma), DPX (Sigma), AdipoR1 shRNA plasmid (Qiagen), phosphatase inhibitor, protease inhibitor cocktail (GenDEPOT), a protein assay kit (Bio-Rad), skim milk (BD Difco), O.C.T. compound (Sakura), ECL solution (ATTO), and fluorescence mounting medium (Agilent Technologies) were purchased from the indicated manufacturers.
Phosphatase inhibitor
Manufactured by Bio-Rad
Sourced in United States
Phosphatase inhibitors are laboratory reagents used to prevent the activity of phosphatase enzymes. Phosphatases are responsible for removing phosphate groups from proteins, which can impact various cellular processes. These inhibitors help maintain the phosphorylation state of proteins, allowing for the study of signaling pathways and other biological mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Phenyl methyl sulfonyl fluoride (pmsf), by Bio-Rad (4 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Anti iba1, by Fujifilm (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Insulin, by Merck Group (1 mentions)
Dibutylphthalate polystyrene xylene (dpx), by Merck Group (1 mentions)
Anti neun, by Merck Group (1 mentions)
Anti p ampk, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti ampk, by Santa Cruz Biotechnology (1 mentions)
Anti adiponectin receptor 1, by Abcam (1 mentions)
Branched pei, by Merck Group (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti tau, by Santa Cruz Biotechnology (1 mentions)
D glucose solution, by Merck Group (1 mentions)
Anti aβ, by Santa Cruz Biotechnology (1 mentions)
Anti p tau, by Santa Cruz Biotechnology (1 mentions)
Anti tnf α, by Santa Cruz Biotechnology (1 mentions)
Anti p akt, by Santa Cruz Biotechnology (1 mentions)
Anti caspase 3, by Santa Cruz Biotechnology (1 mentions)
6 protocols using phosphatase inhibitor
1
Multimodal Analysis of Neuroinflammation
2
Western Blot Analysis of Retinal Proteins
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Protein concentrations of the retinal tissues homogenized in the radioimmunoprecipitation assay buffer containing phenylmethylsulfonyl fluoride and phosphatase inhibitor (Bio-Rad, Hercules, CA, USA) were determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Proteins (10-30 µg/sample) were denatured in the Laemmli buffer for 10 minutes at 70°C (or room temperature for opsins), separated in a polyacrylamide gel, and transferred to an Immobilon-P membrane (Millipore, Burlington, MA, USA) by electrophoresis. The membrane was incubated in blocking buffer, primary antibody, and the horseradish peroxidase–conjugated secondary antibody against mouse or rabbit IgG (7076 and 7074; Cell Signaling Technology, Danvers, MA, USA). Immunoblots were visualized with the ECL Prime Western blotting detection reagent and ImageQuant LAS 4000. The primary antibodies used are for RIPK1 (610458; BD Bioscience, San Jose, CA, USA); MLKL (SAB2103620), M-opsin (AB5405), Tubulin (T2200) (Millipore); RIPK3 (95702), phospho-MLKL (pMLKL, 37333) (Cell Signaling Technology); p62 (sc-48402), S-opsin (sc-14363) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), or rhodopsin (ab5417; Abcam, Cambridge, MA, USA).
3
Protein Extraction and Western Blot Analysis
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After 72 h of transfection, cells were washed with a pre-chilled PBS solution three times; then, the were mixed with RIPA lysis buffer (Beyotime Institute of Biotechnology) containing PMSF (Bio-Rad Laboratories, Inc.) and phosphatase inhibitors (Bio-Rad Laboratories, Inc.) (100:1:1) to the cracks for 30 min. The bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology) was used to detect the total protein content in the cell supernatant, and the upper liquid was further allocated to the ready liquid containing 3 ng/µl protein. Proteins were separated by SDS-PAGE and then transferred onto PVDF membranes (Institute of Biotechnology). The membranes were then blocked with Tween-20 (TBST) in TBS at 37 °C for 1 h. Next, the PVDF membranes were incubated with the appropriate primary antibodies and incubated overnight at 4 °C. Then, the PVDF membranes were incubated with anti-rabbit or anti-mouse IgG secondary antibodies at 37 °C for 1 h. An electrochemiluminescence kit was used to visualize proteins on the PVDF membranes in a dark room, and images of the stained proteins were captured using a ChemiDoc™ Molecular Imager. Each experiment was repeated at least three times.
4
Western Blot Analysis of Glioma Proteins
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Protein was extracted from glioma cells using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) containing PMSF (Bio-Rad Laboratories, Inc.) and Phosphatase inhibitors (Bio-Rad Laboratories, Inc.) (100:1:1). Then proteins were separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane (Beyotime Institute of Biotechnology). Next, the indicated primary antibodies were used for immuno-detection with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG antibodies and electrochemiluminescence chromogenic kit (Beyotime Institute of Biotechnology). The antibodies used for western blot were showed in the Supplementary Table 4 . The experiments were repeated at least three times.
5
Protein Extraction and Western Blot Analysis
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The total protein was harvested with RIPA lysis buffer (Beyotime Institute of Biotechnology) containing PMSF (Bio‐Rad Laboratories, Inc.) and phosphatase inhibitors (Bio‐Rad Laboratories, Inc.) (100:1:1). This experiment was performed as described in a previous study.[4 ] And the primary antibodies included N‐cadherin, E‐cadherin, vimentin, snail, slug, β‐tubulin, MYH10, and MYH9 (Table S3 , Supporting Information). The experiments were repeated at least three times.
6
Protein Extraction and Western Blotting
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The SUNE-1 and HONE-1 cells were washed three times with ice-cold PBS and total protein was harvested using RIPA lysis buffer (Beyotime Institute of Biotechnology) containing PMSF (Bio-Rad Laboratories, Inc.) and Phosphatase inhibitors (Bio-Rad Laboratories, Inc.) (100:1:1). This assay was performed as described in a previous study [25] . The primary antibodies included MYL9 (15354-1-AP, Proteintech, Rabbit), N-cadherin (66219-1-Ig, Proteintech, Mouse), E-cadherin (60335-1-Ig, Proteintech, Mouse), Vimentin (10366-1-AP, Proteintech, Rabbit), ABCB1 (22336-1-AP, Proteintech, Rabbit), ABCG2 (27286-1-AP, Proteintech, Rabbit), and GAPDH (CW0100M, CWBIO, Mouse). The following secondary antibodies were used: HRP-conjugated goat anti-rabbit (cat. no. 6401-05; dilution 1:5000; Biovision, Inc.) and HRPconjugated anti-mouse IgG antibody (cat. no. 6402-05; dilution 1:5000; Biovision, Inc.).
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