Cells were lysed and sonicated in radioimmunoprecipitation (RIPA) buffer with protease inhibitor mixture, PMSF, and sodium orthovanadate. Protein concentration was measured by BCA protein assay (Thermo scientific, Rockford, IL, USA). The samples were resolved by SDS-PAGE and transferred to nitrocellulose membrane and blotted with specific antibodies: anti-p58IPK, anti-p-NF-κB, anti-NF-κB, anti-p-p38, anti-p38, anti-p-JNK, anti-JNK, (Cell Signaling Technology, Boston, MA, USA), anti-p-PKR, anti-PKR, anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-IL-1β (R&D Systems, Inc., Minneapolis, MN, USA), anti-caspase-1 (p-20), and anti-NLRP3 (AdipoGen, Inc, San Diego, CA, USA). The immunoblots were developed using chemiluminescence (SuperSignal West Dura Extended Duration Substrate, Thermo Scientific, USA) and visualized under Chemidoc MP Imaging System (Bio-Rad, Hercules, CA, USA).
Anti p58ipk
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-p58IPK is a primary antibody that recognizes the p58IPK protein. p58IPK is a cellular protein that plays a role in the regulation of protein synthesis. This antibody can be used to detect and study the p58IPK protein in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Santa Cruz Biotechnology (2 mentions)
Anti p eif2α, by Cell Signaling Technology (2 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Anti p p38, by Cell Signaling Technology (1 mentions)
Anti p38, by Cell Signaling Technology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Anti pkr, by Santa Cruz Biotechnology (1 mentions)
Anti p jnk, by Cell Signaling Technology (1 mentions)
Anti il 1β, by R&D Systems (1 mentions)
Anti nf κb, by Cell Signaling Technology (1 mentions)
Anti jnk, by Cell Signaling Technology (1 mentions)
Anti p nf κb, by Cell Signaling Technology (1 mentions)
Anti nlrp3, by Adipogen (1 mentions)
Anti caspase 1 p20, by Adipogen (1 mentions)
Clarity and clarity max ecl western blotting substrate, by Bio-Rad (1 mentions)
Anti β actin, by Abcam (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Sodium orthovanadate, by Santa Cruz Biotechnology (1 mentions)
4 protocols using anti p58ipk
1
Western Blot Analysis of Inflammatory Signaling
2
Western Blot Analysis of p58IPK in Murine Cells
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Mouse retinal tissue or BMDMs were lysed with radio immune precipitation assay (RIPA) buffer with a protease inhibitor mixture, PMSF, and sodium orthovanadate (Santa Cruz Biotechnology). Protein concentration was quantified using the PierceTM BCA protein assay kit (ThermoFisher). Samples were loaded into a 10% SDS-PAGE gel, and transferred to nitrocellulose membrane for immunoblotting using anti-p58IPK (Cell Signaling Technology, Danvers, MA, USA, C56E7, 1:1000) and anti-β-actin (Abcam, Cambridge, United Kingdom, ab8226, 1:10000) antibody. After incubation with HRP-conjugated secondary antibodies, membranes were developed with Clarity and Clarity Max ECL Western Blotting Substrate (Bio-Rad, CA, USA) using Chemi-Doc MP Imaging System (Bio-Rad, CA, USA). The bands were semi-quantified by densitometry using Image J software.
3
Quantitative analysis of UPR markers
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Cells were lysed in M-PER mammalian protein extraction reagent containing Halt Protease (Pierce Biotechnology, Rockford, lL) following the manufacturer’s recommendations. Twenty micrograms of proteins from these cell free extracts were analyzed on SDS-PAGE (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membrane using Trans-Blot Turbo transfer system (Bio-Rad, Hercules, CA). Membranes were probed with rabbit anti-BiP, anti-PDI, anti-p-PERK, anti-eIF2α, anti-P-eIF2α, anti-P58IPK (Cell Signaling, Boston, MA, USA), mouse anti-ATF-6 (Pierce Biotechnology; Rockford, lL, USA), and rabbit anti-XBP1 (Abcam, Cambridge, MA, USA). Antigen specific bands were detected by incubation of membranes using HRP-conjugated anti-rabbit or anti-mouse antibodies followed by incubation with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, lL, USA) and image analysis by ImageQuant LAS4000 (GE Heathcare Bio-Sciences, Pittsburgh, PA, USA).
4
Western Blot Analysis of Stress Markers
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Radioimmuno precipitation assay (RIPA) buffer with protease inhibitor mixture, PMSF, and sodium orthovanadate (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to extract the proteins from cells or tissues. A BCA protein assay kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA) was used to measure protein concentration. Twenty-five micrograms of protein were resolved by SDS-PAGE and blotted with specific antibodies: anti-XBP1, anti-ATF4 (CREB2; Santa Cruz Biotechnology); anti-cleaved caspase-3, anti-ZO-1, anti-occludin (Invitrogen, Carlsbad, CA, USA), anti-p-eIF2α, anti-CHOP, anti-p58IPK (Cell Signaling Technology, Boston, MA, USA); or anti-KDEL, anti-ATF6 (Abcam, Cambridge, MA, USA). The same membrane was stripped and reblotted with an anti-β-actin antibody (Abcam) as loading control. After incubation with peroxidase-labeled secondary antibodies (Vector Laboratories, Inc., Burlingame, CA, USA), membranes were developed with SuperSignal West Dura Chemiluminescent Substrate (Thermo Fisher Scientific, Inc., Rockford, IL, USA). Protein bands were quantified by densitometry, normalized to β-actin (loading control).
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