Cell lysates were prepared in RIPA lysis buffer containing phenylmethylsulfonyl fluoride (PMSF). The protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), then were transferred onto the polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked with 5% non-fat dry milk for 1 h. Primary rabbit antibodies include anti-Bip (1:1000, Cell Signaling), anti-CHOP (1:1000, Cell Signaling), anti-PERK (1:1000, Cell Signaling), phospho-PERK (1:1000, Invitrogen), IRE1-α (1:1000, Cell Signaling), p-IRE1-α (1:1000; Invitrogen), splicing XBP1 (1:1000, Cell Signaling), ATF6 (1:1000, Cell Signaling). HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were used and the chemiluminescent signal was detected by using electrochemiluminescence (ECL) reagents (Invitrogen).
Phospho perk
Manufactured by Thermo Fisher Scientific
Sourced in United States
Phospho-PERK is a laboratory equipment product that detects and measures the phosphorylation of the PERK (Protein Kinase R-like Endoplasmic Reticulum Kinase) protein. PERK is a key regulator of the unfolded protein response, a cellular mechanism that responds to endoplasmic reticulum stress. The Phospho-PERK product enables researchers to study PERK activation and its role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
P ire1α, by Thermo Fisher Scientific (3 mentions)
Splicing xbp1, by Cell Signaling Technology (3 mentions)
Anti p erk, by Cell Signaling Technology (2 mentions)
Anti chop, by Cell Signaling Technology (2 mentions)
Ire1α, by Cell Signaling Technology (2 mentions)
Rc dc assay, by Bio-Rad (2 mentions)
P akt serine 473, by Cell Signaling Technology (2 mentions)
Gapdh, by Thermo Fisher Scientific (2 mentions)
Phospho eif2α, by Cell Signaling Technology (2 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Anti bip, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti mouse cd4, by Cell Signaling Technology (1 mentions)
Image studio 2, by LI COR (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Perm wash buffer, by BD (1 mentions)
8 protocols using phospho perk
1
Quantifying ER Stress Pathway Activation
2
Western Blot Analysis of UPR Markers
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Cell lysates were prepared in RIPA lysis buffer containing phenylmethylsulfonyl fluoride (PMSF). The protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then were transferred onto the polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked with 5% nonfat dry milk for 1 h. Primary rabbit antibodies include anti-β-actin (1 : 1000, Cell Signaling), anti-CHOP (1 : 1000, Cell Signaling), anti-PERK (1 : 1000, Cell Signaling), phospho-PERK (1 : 1000, Invitrogen), IRE1-α (1 : 1000, Cell Signaling), p-IRE1-α (1 : 1000; Invitrogen), and splicing XBP1 (1 : 1000, Cell Signaling). HRP-conjugated anti-rabbit secondary antibodies were used, and the chemiluminescent signal was detected by using electrochemiluminescence (ECL) reagents.
3
Immunohistochemical Analysis of Tumor Microenvironment
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The mice were sacrificed 48 hours after irradiation. Tumor tissues and spleen tissues were fixed in paraffin, imbedded, and cut for 4 mm sections. Tumor sections were incubated with primary antibodies, including anti-mouse CD4 (1 : 100, Cell Signaling), CD8 (1 : 100, Cell Signaling), CD11c (1 : 100, Cell Signaling), FoxP3 (1 : 100, Cell Signaling), CHOP (1 : 100, Cell Signaling), phospho-PERK (1 : 100, Invitrogen), p-IRE1-α (1 : 100; Invitrogen), and splicing XBP1 (1 : 100, Cell Signaling) antibodies, at 4°C overnight, and biotin-labeled secondary antibody for 30 min at 37°C. The final signal was developed using the 3,3′-diaminobenzidine (DAB) substrate, and the sections were observed under optical microscope. The percentage of positive cells was calculated as number of positive (brown) cells/total number of cells × 100 in 9 randomly selected fields (400×).
4
Evaluation of ER Stress Response
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Infected mammalian cells were lysed in radioimmunoprecipitation assay buffer with the addition of protease (Roche cOmplete), and phosphatase inhibitors (GB Sciences). Protein levels of lysates were determined using the BioRad DC/RC assay. Equal amounts of protein lysate were boiled with SDS load buffer. Equal amounts of protein were loaded and immunoblotting with, Thermo GAPDH antibody was used as a loading control. Antibodies used in assay are as follows: CHOP (Thermo MA1-250; 1:2,000), BiP (Protein Tech Group 11587-1-AP; 1:5,000), Phospho-Perk (Thermo MA5-15033; 1:1,000), GAPDH (Thermo MA5-15738; 1:10,000), PDI (Enzo ADI-SPA-891; 1:5,000), P115 (Lab generated; 1:2,500), p-AKT Serine 473 (Cell Signaling 9271S; 1:1,000).
5
Protein Isolation and Western Blot Analysis
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For protein isolation, BMDMs were seeded (1x 106 cells per well of a 6-well plate) and infected in triplicate as describe. Triplicate wells of macrophages were lysed in a 300 μl total of radioimmunoprecipitation assay (RIPA) buffer (50 mM TrisHCl pH8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Insoluble debris was removed by centrifugation. Protein concentrations were determined using Pierce BCA Protein Assay Kit (Thermo Scientific). Equivalent amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were incubated with antibodies per manufacturer’s suggestions. Blots were imaged on an Odyssey CLx and analyzed using ImageStudio2.1 (Licor). The following primary antibodies were used: phospho-PERK (ThermoFisher Scientific G.305.4), phospho-eIF2α (Cell Signaling Technology 9721), α-tubulin (Santa Cruz Biotechnology YOL1/24; sc-53030), ATF4 (Cell Signaling Technology D4B8; 11815), CHOP (Cell Signaling Technology L633F7; 2895), TRIB3 (Calbiochem ST1032), phospho-Akt (Cell Signaling Technology C313E5E; 2965), Akt (Cell Signaling Technology 40D4; 2920). Images were processed with Adobe Photoshop, which was utilized on occasion to change the order of lanes in the image to group like samples (e.g. lytic strains or non-lytic strains) together.
6
Multiparameter Flow Cytometry of Cellular Stress Responses
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At first, cells were incubated with 100 µl of CD16/32 Fc block diluted 1/50 in PBS for 15 min at 4°C. After wash, cells were stained in PBS with CD45.2-APC/Cy7, CD45.1-BV421, CD11b-BUV737, Ly- 6G/C-PE/Cy7 mouse antibodies (Table 7 ), and a LIVE/DEAD™ fixable dead cell stain (Thermo Fisher Scientific; Cat# L34961) in the dark for 30 min at RT. Then cells were washed, pelleted, and fixed with Cytofix/Cytoperm Fixation & Permeabilization (BD Biosciences). Samples were washed twice with 1X BD Perm/Wash™ Buffer (BD Biosciences) + 2% FBS (WashB), being pelleted at 1,000 g x 5 min at 4°C. Then cells were incubated overnight at 4°C with the following mouse primary antibodies: ATF-4 (Cell Signaling Technology), AFT-6 (Cell Signaling Technology), BiP (Cell Signaling Technology), CHOP (Cell Signaling Technology), phospho-eIF2α (Cell Signaling Technology), or phospho-PERK (Thermo Fisher Scientific). The next day, samples were washed four times and pelleted. Cells were incubated with a donkey anti-rabbit AF488 secondary antibody (Thermo fisher Scientific) for 1-hour at 4°C, after which cells were washed four times and pelleted. Samples were then resuspended in FACS buffer and analyzed on an LSRII (BD Biosciences).
7
Immunoblotting Analysis of UPR Markers
8
Western Blot Analysis of UPR Proteins
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Primary antibodies were used as follows: rabbit anti-ATF-6, WT-1, LC3B and X-BP1 antibodies (Abcam); BiP, CHOP, caspase-12, caspase-3, LC3 A/B I/II and GAPDH antibodies (Cell Signaling Technology, MA, USA); phospho-IRE1α (Novus Biologicals, CO, USA); phospho-PERK (Thermo Fisher Scientific, MA, USA). Kidney cortex or cultured podocytes were lysed in lysis buffer with a sonicator and centrifuged at 14 000 × g for 5 min at 4 °C. In all, 40 μg of total protein samples were separated by 8-12% gel electrophoresis and electrotransferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% bovine serum albumin (BSA) for 1 h and incubated with primary antibodies overnight at 4 °C. Then the membranes were washed in Tris-buffered NaCl solution with Tween 20 (TBST, containing 0.1% Tween 20), and then incubated with horseradish peroxidase-conjugated secondary antibodies in 5% BSA for 1 h. Density of the corresponding bands was measured by Bio-Rad VersDoc imaging system using chemiluminescence detection reagents (Thermo Fisher Scientific). The data were analyzed with Bio-Rad Quantity One software (Bio-Rad, CA, USA), and corrected by reference to the value of GAPDH.
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