The gastrocnemius muscle was homogenized in lysis buffer containing 50 mM HEPES, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamide, 1 mM Na3VO4, 1 mM dithiothreitol (DTT), 5 mM MgCl2, 1% NP40, 10% glycerol, aprotinin, leupeptin, and pepstatin A. The homogenate was centrifuged at 20000×g for 15 min at 4℃. The protein concentration of the supernatant was measured using the Bradford assay. SDS-PAGE was used to separate 80 µg of protein that was then blotted on a PVDF membrane. The membrane was blocked with 0.1% Tween in tris-buffered saline (TBST) with 5% skimmed milk for 1 h at room temperature. Primary antibodies (PERK, phosphorylated PERK, BiP, CHOP, and GAPDH [PERK, phosphorylated PERK, BiP, and CHOP from Cell Signaling, Danvers, MA, USA; GAPDH from Santa Cruz Biotechnology, Santa Cruz, CA, USA]) were diluted 1:700 in TBST with 5% bovine serum albumin, and were incubated overnight at 4℃. The membranes were incubated with goat anti-rabbit/mouse IgG HRP antibodies (Bio-Rad, Hercules, CA, USA) for 1 h at room temperature then diluted 1:2000 in TBST with 5% skim milk. The membrane was examined using electrochemiluminescence. In this study, GAPDH was used as a control to ensure equal protein loading on the gel and the blots were quantified using MultiGauge software.
Phosphorylated perk
Manufactured by Cell Signaling Technology
Sourced in United States
Phosphorylated PERK is a protein reagent used in research applications. It is a post-translationally modified form of the protein PERK (Protein Kinase R-like Endoplasmic Reticulum Kinase), which plays a role in the unfolded protein response. This phosphorylated form of PERK can be used to study signaling pathways and cellular stress responses.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (2 mentions)
Eif2α, by Cell Signaling Technology (2 mentions)
Hrp conjugated goat anti rabbit, by Cell Signaling Technology (2 mentions)
Phosphorylated eif2α, by Cell Signaling Technology (2 mentions)
Luminata crescendo western hrp substrate, by Merck Group (2 mentions)
Las 4000 imager, by Fujifilm (2 mentions)
Cell counting kit 8 cck 8, by MedChemExpress (1 mentions)
C fos, by Cell Signaling Technology (1 mentions)
V atpase d2, by Cell Signaling Technology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Phenylmethanesulfonyl fluoride, by Merck Group (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Hrp conjugated donkey anti rabbit and anti mouse immunoglobulin ig g secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Epigallocatechin gallate (egcg), by MP Biomedicals (1 mentions)
Cell culture media, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Perilipin, by Cell Signaling Technology (1 mentions)
Phosphorylated ampk, by Cell Signaling Technology (1 mentions)
11 protocols using phosphorylated perk
1
Western Blot Analysis of Endoplasmic Reticulum Stress
2
Osteoclastogenesis Assay Protocol
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Ppg (HPLC98%) was provided by the Chengdu Must Biological Technology Co., Ltd. (China) for this study. Fetal bovine serum (FBS), penicillin-streptomycin (PS) solution, and alpha-modified minimum essential medium (α-MEM) were supplied by Gibco (Thermo Fisher Scientific, United States). Dimethyl sulfoxide (DMSO) was used to dissolve Ppg to a storage concentration of 100 mM before diluting Ppg to different concentrations by adding α-MEM. Moreover, Cell Counting Kit-8 (CCK-8) was supplied by MedChemExpress (MCE, China). Recombinant M-CSF and RANKL mice were supplied by R&D Systems (USA). Specific primary antibodies, such as p-P65, P65, phosphorylated (p)-ERK, ERK, p-JNK (c-Jun N-terminal kinase), JNK, p-P38, P38, β-actin, V-ATPase d2, and c-Fos, as well as secondary antibodies, such as rabbit and mouse, were supplied by Cell Signaling Technology (USA). Furthermore, Abcam (UK) provided the antibodies against CTSK, IκBα, and NFATc1.
3
Western Blotting for Protein Analysis
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Western blotting analyses were performed as previously described (Simar et al., 2014 (link); Wang et al., 2015 (link)). Total soluble proteins were extracted with radio-immunoprecipitation assay lyses buffer (Applygen Technologies Inc., Beijing, China) containing phenylmethane sulfonyl fluoride (Sigma-Aldrich). Protein concentrations were determined with the BCA method; equal amounts of total protein were loaded on 10% denatured polyacrylamide gels and transferred electrophoretically to nitrocellulose membranes (Hybond). The blotted membranes were blocked with 5% (w/v) nonfat derived milk in Tris-buffered saline for 1 h at room temperature and then were incubated with primary antibodies against β-actin, ERK (extracellular signal-regulated kinase), STAT3 (signal transducer and activator of transcription), phosphorylated (p)-ERK, and p-STAT3 (1:1,500, Cell Signaling Technology) for 16 h at 4°C. After incubation with IRDye 800 CW-conjugated goat anti-rabbit IgG antibodies, the labeled bands were visualized and quantified with an Odyssey Infrared Imaging system (LI-COR. Bioscience).
4
Immunoblotting of Signaling Proteins
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Sodium dodecyl sulfate (SDS) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Corp (St Louis, MO, USA). Cell culture media was obtained from Life Technologies Corp (Carlsbad, CA, USA). Electrophoresis reagents were purchased from Bio-Rad Laboratories (Hercules, CA, USA). The HyGLO™ Chemiluminescent HRP (horseradish peroxidase) Antibody Detection Reagents were from Denville Scientific Inc. (Metuchen, NJ, USA). Micro bicinchoninic acid (BCA) protein assay reagents were from Pierce (Micro BCA™ Protein Assay Kit; Thermo Fisher Scientific, Waltham, MA, USA). The polyclonal antibodies against extracellular signal-regulated kinase (ERK), phosphorylated (P)-ERK, c-Jun N-terminal kinase (JNK), P-JNK, P38, and P-P38 were all purchased from Cell Signaling Technology, Inc (Danvers, MA, USA). HRP-conjugated donkey anti-rabbit and anti-mouse immunoglobulin (Ig) G secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). EGCG was from MP Biomedicals (Santa Ana, CA, USA). All other reagents were from Sigma-Aldrich Corp.
5
Molecular Mechanisms of AMPK Regulation
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TNF-α and 2-aminopurine (2-AP) were purchased from Sigma Chemical (St. Louis, MO, USA). AICAR and compound C (CC) were purchased from Calbiochem (San Diego, CA, USA). Antibodies against AMPK, phosphorylated-AMPK, β-actin, perilipin, hormone sensitive lipase (HSL), eIF2α, phosphosphorylated-eIF2α, PERK, and phosphorylated-PERK were purchased from Cell Signaling (Beverly, MA, USA).
6
Coronary Artery Protein Expression Analysis
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Freshly isolated coronary arteries from all of the groups were immediately frozen in liquid nitrogen and then homogenized in ice-cold lysis buffer, as previously described35 (link). Western blot analysis was performed for BiP (binding immunoglobulin protein; 1:1000 dilution; Cell signaling, Danvers, MA, USA), phosphorylated IRE1, XBP-1 (1:1000 dilution; Cell signaling, Danvers, MA, USA), phosphorylated-eIF2α (1:1000 dilution; Abcam, Cambridge, MA, USA), cleaved ATF6 (1:1000 dilution; Cell signaling, Danvers, MA USA), CHOP (1:1000 dilution; Cell signaling, Danvers, MA, USA), and phosphorylated PERK (1:1000; Cell Signaling, Danvers, MA, USA), and phosphorylated 20 KDa myosin light chain (MLC20, 1:1000; Cell Signaling, Danvers, MA, USA). Blots were stripped and then reprobed with the β-actin antibody (1:2000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA) to verify the equal loading between the samples.
7
Comprehensive Western Blot Analysis
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Western blot analysis was performed according to a previously described standard method23 (link). Primary antibodies against the following targets were used for the Western blot analyses: HER2 (#2165, diluted at 1: 500, Cell Signaling Technology), Cyclin B1 (#4138, 1: 1000, Cell Signaling Technology), Cyclin D1 (#2922, 1: 1000, Cell Signaling Technology), HMGB1 (ab79823, 1: 10000, Abcam), GSDMB (A7474, 1: 1000, ABclonal), caspase-1 (A18646, 1: 1000, ABclonal), NLRP3 (A12694, 1: 1000, ABclonal), ERK (#9102, 1: 1000, Cell Signaling Technology), phosphorylated (p)-ERK (#4370, 1: 1000, Cell Signaling Technology), AKT (#9272, 1: 1000, Cell Signaling Technology), phosphorylated (p)-AKT (#4060, diluted at 1: 1000, Cell Signaling Technology), Nrf2 (#12721, 1: 1000, Cell Signaling Technology), β-actin (used as the loading control; sc-47778, Santa Cruz Biotechnology), GAPDH (used as the loading control; sc-47724, Santa Cruz Biotechnology), and β-tubulin (used as the loading control; #2128, Cell Signaling Technology).
8
Antibody and Small Molecule Profiling for ER Stress Response
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Antibodies including HT-7 (catalog no.: MN1000; Invitrogen), AT8 (catalog no.: MN1020; Invitrogen), YFP (catalog no.: ab6556; Abcam), HSP90 (catalog no.: ab13492; Abcam), lamin A/C (catalog no.: 2032; Cell Signaling Technology), GAPDH (catalog no.: ab8245; Abcam), T-PERK (catalog no.: 3192; Cell Signaling Technology), phosphorylated PERK (catalog no.: 3179; Cell Signaling Technology), eIF2α (catalog no.: 5324; Cell Signaling Technology), p-eIF2α (catalog no.: 5324; Cell Signaling Technology) were pretested to detect the targeted proteins. Small molecules included GSK2656157 (catalog no.: 9466-5; BioVision), GSK2606414 (catalog no.: S7307; Selleckchem), salubrinal (catalog no.: S2923; Selleckchem), ISRIB (catalog no.: S7400; Selleckchem); Ceapin-A7 (catalog no.: SML2330; Sigma–Aldrich), 4u8C (catalog no.: CAS14003-96-4; Calbiochem), and AA147 (product no.: 6538059; ChemBridge) were prepared in dimethyl sulfoxide following the manufacturer’s instruction and stored at −80 °C as stock solution. ER stress–inducing chemical, thapsigargin, was dissolved in dimethyl sulfoxide and added to the cell culture media at a concentration of 300 nM. The working solutions were freshly prepared with −80 °C stock solution.
9
Western Blot Analysis of PERK and eIF2α Phosphorylation
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Lysates were separated by SDS-PAGE with 10% polyacrylamide gels and transferred to PVDF membranes. Membranes were blocked in 5% BSA in TBST for 1 h, and probed with antibodies to phosphorylated PERK (Cell Signaling Technology; 1:200), phosphorylated eIF2α (Cell Signaling Technology; 1:1,000), total PERK (Cell Signaling Technology; 1:1,000), and total eIF2α (Cell Signaling Technology; 1:1,000) overnight at 4°C. Membranes were washed and incubated with HRP-conjugated goat anti-rabbit (Cell Signaling Technology; 1:2,000) for 1 h. Chemiluminescence was detected with Luminata Crescendo Western HRP Substrate (Millipore) and analyzed with Fujifilm LAS-4000 imager and Multi Gage Software (Fujifilm Life Science).
10
Immunohistochemical Analysis of Lung Tissue
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Expression levels of ORMDL3, MMP-9 and ERK in the formalin-fixed, paraffin-embedded lung tissue sections were assessed by immunohistochemical staining. Samples were incubated with antibodies specific to ORMDL3 (dilution, 1:200; cat. no. ab107639; Abcam, Cambridge, UK), ERK (dilution, 1:200; cat. no. 9102; Cell Signaling Technology, Inc., Danvers, MA, USA), phosphorylated (p)-ERK (dilution, 1:200; cat. no. 9103; Cell Signaling Technology, Inc.) and MMP-9 (dilution, 1:200; cat. no. AB19016; Merck KGaA) overnight, at 4°C, then staining was detected with biotinylated secondary antibodies (goat anti-rabbit horseradish peroxidase-IgG secondary antibodies; dilution, 1:2,000; cat. no. SP-9001; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) and DAB reagent (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) at 37°C in the incubator for 30 min. A total of 10 fields of each specimen were reviewed, in 10 random visual fields, and staining was assessed using ImageJ software v.10.2 (https://imagej.nih.gov/ij/ ).
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