Western blotting was performed as described previously [20 (link)]. Whole-cell lysates were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. After incubation with the appropriate antibodies, bound antibodies were detected using chemiluminescent HRP substrate (Millipore, St. Louis, MO, USA). The blots were exposed and quantified using an Alliance Mini 4M (UVITEC Cambridge, Cambridge, UK). The primary antibodies were as follows: Anti-MFN1/2, anti-OPA1, anti-DRP1, anti-MFF, anti-GRP78/BiP, anti-CHOP, anti-phospho-eIF2a, anti-caspase-3 (Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). The secondary antibodies were anti-rabbit IgG-HRP (Cell Signaling Technology) and anti-mouse-IgG-HRP (Calbiochem, Darmstadt, Germany).
Anti phospho eif2a
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-eIF2a is a laboratory reagent that specifically detects the phosphorylated form of the eIF2a protein. eIF2a is a subunit of the eukaryotic translation initiation factor 2 complex, and its phosphorylation is a key regulatory mechanism in the cellular stress response.
Automatically generated - may contain errors
Lab products found in correlation
Anti eif2a, by Cell Signaling Technology (3 mentions)
Anti chop, by Cell Signaling Technology (2 mentions)
Anti lc3, by Cell Signaling Technology (2 mentions)
Anti p70s6k, by Cell Signaling Technology (2 mentions)
Anti phospho p70s6k, by Cell Signaling Technology (2 mentions)
Anti phospho mtor, by Cell Signaling Technology (2 mentions)
Anti atf4, by Cell Signaling Technology (2 mentions)
Anti mtor, by Cell Signaling Technology (2 mentions)
Anti phospho ampk, by Cell Signaling Technology (2 mentions)
Anti ampk, by Cell Signaling Technology (2 mentions)
Anti p erk, by Cell Signaling Technology (2 mentions)
Anti drp1, by Cell Signaling Technology (1 mentions)
Anti grp78 bip, by Cell Signaling Technology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Chemiluminescent hrp substrate, by Merck Group (1 mentions)
Anti opa1, by Cell Signaling Technology (1 mentions)
Anti mff, by Cell Signaling Technology (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
Alliance mini 4m, by Uvitec (1 mentions)
7 protocols using anti phospho eif2a
1
Western Blotting Analysis of Mitochondrial Dynamics
2
Regulation of MITF Expression by α-Mangostin and ATRA
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Western Blot analysis was performed as described previously [14 (link)]. The following primary antibodies were used: anti-ERK, anti-phospho-ERK (Thr202/Tyr204), anti-AKT, anti-phospho- AKT (Thr473), anti-Mcl-1, anti-beta-actin, anti-phospho-eIF2a, anti-eIF2a, anti-LC3, anti-Atg5, anti-MITF (Cell Signaling Technology). UACC257 cells were cultured in Vitamin D deficient medium for more than one week, and then treated with different concentration of α-Mangostin (5 μM or 0.5 μM), ATRA (5 μM or 0.5 μM) or their combination for 8 hours. The expression level of MITF was detected by western blot. ChemiDoc XRS system (Bio-Rad) was used for imaging.
3
Immunohistochemical analysis of inner ear
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The inner ears were fixed in 4% paraformaldehyde (PFA) overnight and then decalcified in 10% ethylenediaminetetraacetic acid (EDTA). After being dehydrated in sucrose and embedded in tissue OCT freeze medium at −20 °C, 6 μm continuous sections were cut. These sections were permeabilized in 0.5% Triton X-100 for 30 min, blocked in 3% BSA for 2 h at room temperature, and then incubated overnight at 4 °C with 1:200 diluted primary antibodies: anti-CDH23 (Santa Cruz sc-26338), anti-BiP (Cell Signaling 3177), anti-CHOP (Santa Cruz sc-575), anti-eIF2a (Cell Signaling 9722) and anti-phospho-eIF2a (Cell Signaling 3597). Secondary antibodies with 1:400 dilution (donkey anti-rabbit IgG, Invitrogen A31573; rabbit anti-goat IgG, Invitrogen A11078) were added for 1 h; the sections were then counter-stained with DAPI. The negative controls (with no primary antibodies added) showed no staining.
4
Synthesis and Characterization of Organometallic Compounds
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NiPT, PtPT, PdPT, T-AuPT, Au(PPh3)PT, CuPT, and AgDT were synthesized in our laboratory and stored as a 10 mM stock solution in dimethyl sulfoxide (DMSO) at −20°C. Auranofin was purchased from Enzo Life Sciences International, Inc. (Plymouth Meeting, PA) and dissolved in DMSO as a 10 mM, stored at −20°C. Bortezomib (Cell Signaling Technology, Beverly, MA, USA) HA–ubiquitin–vinyl sulfone (HA-Ub-VS), and Ub-AMC (BostonBiochem, Cambridge, MA, USA). Antibodies were purchased from following sources: anti-AMPK, anti- phospho-AMPK, anti-mTOR, anti-phospho-mTOR, anti-P70S6K, anti-phospho-P70S6K, anti-S6, anti-phospho-S6, anti-ATF4, anti-CHOP, anti-phospho-eIF2a, anti-LC3, anti-USP14 (D8Q6S), and anti-PARP (Cell Signaling Technology, Beverly, MA, USA); anti-ubiquitin (P4D1) and anti-SQSTM1/P62(Santa Cruz Biotechnology, Santa Cruz, CA); anti-GAPDH and anti-HA-tag (Bioworld Technology, Nanjing, China); and anti-UCHL5 (Abcam, Cambridge, UK). Anti-phospho-USP14 and anti-phospho-UCHL5 were prepared by the Abgent, Inc. Bafilomycin, 3-MA, and E64D were purchased from Enzo Life Sciences (MA, USA). Lipofectamine 2000 (11668–027), goat anti-rabbit Alexa 488 (997773), and opti-MEM medium (22600050) were purchased from the InvitrogenTM.
5
Immunohistochemical Detection of Phospho-eIF2α
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The ovaries were fixed in formalin, embedded in paraffin, and cut into 3-μm-thick sections. The sections were deparaffinized and briefly heated. The sections were then treated with a protein blocking solution (Dako, CA, USA) and incubated with anti-phospho eIF2a (Cell Signaling). After washing with 0.1 M TBS containing 0.01% Tween-20 (TBST), the sections were incubated with anti-rabbit polymer (Dako). Peroxidases bound to the antibody complex were visualized after treatment with a 3, 3′-diaminobenzidine (DAB) chromogen substrate solution (Dako). The DAB reaction was monitored under a microscope to determine the optimal incubation time and stopped with several washes of 0.1 M TBS. The immunolabeled sections were dehydrated in a graded ethanol series, defatted in xylene, and mounted. The sections were examined under a bright field Olympus BX51 microscope and images were acquired using an Olympus DP 70 camera (Olympus, Japan).
6
Quantifying Protein Levels via Western Blot
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Western blot analysis of protein levels was performed on whole cell lysates prepared by lysis in RIPA buffer (Sigma) supplemented with Complete EDTA free proteinase inhibitors (Roche). Pierce Phosphatase Inhibitor Mini Tablets (ThermoFisher) were added to the lysis buffer for the analysis of phosphorylated Perk and eIF2a. The antibodies used for WB analysis are: anti-Pdap1 (Sigma), anti-Tubulin (Abcam), anti-bActin (Sigma), anti-AID (mAID-2, ThermoFisher), anti-phospho-Perk (pThr980, ThermoFisher), anti-Perk (Cell Signaling Technology), anti-phospho-eIF2a (pSer51, Cell Signaling Technology), anti-eIF2a (Santa Cruz), anti-Atf4 (Cell Signaling Technology), and anti-Xbp1-s (Cell Signaling Technology).
7
Genipin-Mediated Tau Aggregation Inhibition
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Genipin was purchased from MedChemExpress (Monmouth Junction, New Jersey, USA). Tau-R3 was obtained from ChinaPeptides (Shanghai, China). Heparin sodium salt was obtained from Aladdin (Shanghai, China). Thio avin T (ThT) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modi ed Eagle medium (DMEM), F12-DMEM, opti-MEM, neurobasal medium, B27 supplement, streptomycin, penicillin, L-glutamine and phosphate buffer solution (PBS) were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was supplied from Biological Industries (Kibbutz Beit Haemek, Israel). The cell counting kit (CCK)-8 and bicinchoninic acid (BCA) protein assay kit were provided by Beyotime (Jiangsu, China). Protease and phosphatase inhibitors were obtained from Bimake (Shanghai, China). The following antibodies were used in this study: anti-Tau, anti-phospho-T231, antiphospho-S396, anti-phospho-S404, anti-CDK5, anti-phospho-GSK-3β (Tyr 216 ), anti-LC3, anti-amyloid precursor protein (APP), anti-Aβ, anti-BACE1, and anti-β-actin (Abcam, Cambridge, UK); anti-p62, anti-Beclin-1, anti-SIRT1, anti-LKB1, anti-phospho-LKB1, anti-AMPK, anti-phospho-AMPK, anti-mTOR, antiphospho-mTOR, anti-p70S6K, anti-phospho-p70S6K, anti-PERK, anti-phospho-PERK, anti-eIF2a, and antiphospho-eIF2a (Cell Signaling Technology, Beverly, MA, USA).
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