Immunoblotting was performed as previously described.24 (link) The following antibodies were used: anti-phosphorylated (p)-ERK, anti-p-Akt, and anti-Ki67 (Cell Signaling, Beverly, MA, USA); anti-cyclin D1, anti-ERK, anti-β-actin, anti-proliferating cell nuclear antigen, and anti-c-myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Rac1 (Upstate Biotechnology, Lake Placid, NY, USA); anti-α-tubulin (Oncogene Research Products, Cambridge, MA, USA); anti-GTP-Rac (NewEast Biosciences, Malvern, PA, USA); and anti-Sur8 produced and purified as previously described.25 (link) Horseradish peroxidase (HRP)-conjugated anti-mouse (Cell Signaling) and anti-rabbit (Bio-Rad Laboratories, Hercules, CA, USA) secondary antibodies were used.
Anti phosphorylated erk
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-phosphorylated ERK product is an antibody that recognizes the phosphorylated form of extracellular signal-regulated kinase (ERK), a key component of the mitogen-activated protein kinase (MAPK) signaling pathway. This antibody can be used to detect and quantify the activated, phosphorylated state of ERK in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti erk, by Cell Signaling Technology (9 mentions)
Anti phosphorylated p38, by Cell Signaling Technology (6 mentions)
Anti p38, by Cell Signaling Technology (4 mentions)
Anti phosphorylated jnk, by Cell Signaling Technology (4 mentions)
Anti jnk, by Cell Signaling Technology (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Anti phosphorylated stat3, by Cell Signaling Technology (3 mentions)
Anti p akt, by Cell Signaling Technology (2 mentions)
Anti total erk, by Cell Signaling Technology (2 mentions)
Axio imager a2 microscope, by Zeiss (2 mentions)
Mowiol 4 88, by Merck Group (2 mentions)
Anti bcl 2, by Cell Signaling Technology (2 mentions)
Anti aif, by Merck Group (2 mentions)
Alexa fluor 568 anti mouse and anti rabbit antibodies, by Thermo Fisher Scientific (2 mentions)
Anti tlr4, by Santa Cruz Biotechnology (2 mentions)
Antiphosphorylated ire1, by Novus Biologicals (2 mentions)
Cell lysis buffer, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti iκbα, by Cell Signaling Technology (2 mentions)
Anti e cadherin, by Cell Signaling Technology (2 mentions)
25 protocols using anti phosphorylated erk
1
Immunoblotting Technique for Protein Analysis
2
Inhibition of EGFR and HSP Signaling
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Gefitinib, SU11274, 17-DMAG, and 17-AAG were obtained from Selleckchem (Houston, TX, USA). All other chemicals were acquired from Sigma Chemical (St. Louis, MO, USA), unless otherwise indicated. Anti-EGFR, anti-total ERK, anti-phosphorylated (p)-ERK, anti-total AKT, and anti-p-AKT antibodies were obtained from Cell Signaling (Danvers, MA, USA). Anti-HSP70 and anti-HSP90 antibodies were obtained from Genetex (Irvine, CA, USA). All other antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
3
Apoptotic Nuclei Detection and Quantification in Photoreceptors
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Eyes were oriented, fixed in Davidson’s fixative (8% Formaldehyde, 31.5% Ethanol, 2 M Acetic Acid), embedded in paraffin and sectioned as previously described37 (link). Apoptotic nuclei were detected by TUNEL kit (fluorescein; Roche) according to the producer’s protocols. Primary antibodies were used as follows: anti-AIF (1:100; Sigma), anti-phosphorylated PERK (1:100, Cell Signaling), anti-RHO (1D4 1:250; Sigma), anti-BAX 6A7 (1:200, BD Biosciences), anti-BCL2 (1:100, Cell Signaling) and anti-calpastatin (1:100, Cell Signaling). Secondary antibodies were Alexa Fluor® 568 anti-mouse and anti-rabbit antibodies (1:1000, Molecular Probes). Slides were mounted with mowiol 4–88 (Sigma) and analyzed at a Zeiss Axio Imager A2 microscope. Quantification of dying cells was performed by counting all TUNEL labeled cells in the photoreceptor cell layer passing through the optic nerve in at least three sections from different animals. The number of photoreceptors was counted by staining of nuclei with DAPI and was used to calculate percentage of dying cells.
4
Standardized Herbal Compound Characterization
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QGHXR (9 g bupleurum root, 9 g scutellaria root, 15 g red sage root, 9 g Carapax Trionycis and 15 g Radix Puerariae), QGR (9 g bupleurum root and 9 g scutellaria root) and HXR (15 g red sage root, 9 g Carapax Trionycis and 15 g Radix Puerariae), were concentrated to 4.75, 1.5 and 3.25 g/ml, respectively, and processed at the Department of Pharmacy, Longhua Hospital (Shanghai, China). In order to ensure the uniformity and stability of herbal products, we performed the HPLC method to detect the contents of four flavones including Puerarin, Baicalin, Baicalein and Wogonin (7 ). Rabbit polyclonal anti-phosphorylated (p)-ERK and mouse anti-rat NF-κB were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and anti-TLR4 was purchased from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA, USA).
5
Apoptosis Quantification in Retinal Photoreceptors
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Eyes were oriented, fixed in Davidson's fixative (8% Formaldehyde, 31.5% Ethanol, 2 M Acetic Acid), embedded in paraffin and sectioned. Apoptotic nuclei were detected by TdTmediated dUTP terminal nick-end labelling kit (TUNEL, fluorescein; Roche) according to the producer's protocols. Primary antibodies were used as follows: anti-AIF (1:100; Sigma), antiphosphorylated-IRE1 (1:200; Novus Biologicals), anti-phosphorylated-PERK (1:100; Cell Signaling). Secondary antibodies were Alexa Fluor ® 568 anti-mouse and anti-rabbit antibodies (1:1000; Molecular Probes). Nuclei were stained with 4′,6-diamidino-2phenylindole (DAPI). Slides were mounted with mowiol 4-88 (Sigma) and analyzed at a Zeiss Axio Imager A2 microscope. Quantification of dying cells was performed by counting all TUNEL labeled cells in the photoreceptor cell layer in at least three entire retinal sections, with a dorsal-ventral orientation, passing through the optic nerve derived from different animals. The number of photoreceptors double labelled with anti-AIF and TUNEL was counted by staining of nuclei with DAPI and was used to calculate percentages of dying cells, as previously published [23] .
6
Retinal Protein Extraction and Analysis
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Retinas were lysed as previously described to obtain nuclear-enriched proteins and total cellular proteins [24] . The purity of enriched lysates was checked by immunoblotting using a nuclear marker (anti-Histone H3 1:3000; Bethyl Laboratories) or a cytosol marker (antipan-actin, 1:3000; Millipore). Equivalent amounts of protein extracts (20 g) were resolved using SDS-PAGE and immunoblottings were performed following standard procedures. The antibodies used for western blotting were: anti-II-spectrin (AA6, 1:2000; Enzo Life) [25] , anti-AIF (1: 1000; Calbiochem) [26] , anti-Caspase7 (1:1000; Cell Signaling), anti-eIF2 (1:1000; Cell Signaling), anti-NRF2 (1:2000; Invitrogen), anti-phosphorylated-IRE1 (1:2000; Novus Biologicals), anti-phosphorylated-PERK (1:1000; Cell Signaling), antiphosphorylated-eIF2 (1:1000; Cell Signaling). Antibodies specificity is shown in Fig. S2.
Each blot analyzed proteins derived from 4 retinas pooled together and 3 independent pools from 3 different litters were used as biological replicates, one representative blot is shown.
The entire blots are shown in Fig. S3.
Each blot analyzed proteins derived from 4 retinas pooled together and 3 independent pools from 3 different litters were used as biological replicates, one representative blot is shown.
The entire blots are shown in Fig. S3.
7
Integrin and ERK Protein Analysis
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Anti- integrin αv antibody was obtained from Millipore (AB1930, 1:1000), anti- integrinβ3 antibody from Abcam (ab75872, 1:1000). Anti-phosphorylated ERK was obtained from Cell Signaling Technology (#9101, 1:1000). Immunoblot analysis was performed as described previously10 (link). Tissue or Cells were lysed in cell lysis buffer (Cell Signaling) with protease inhibitor cocktails (Sigma). Lysates were centrifuged at 15,000 g for 15 min at 4 °C, and supernatants were collected. 20–30 μg of cell lysates were separated by SDS-PAGE gel and transferred to Reinforced NC membrane (Whatman GmbH). The membranes were blocked with 5% skim milk in 0.1% Tween 20/TBS buffer and then incubated with primary antibodies. Blots were probed with horse radish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG (Jackson ImmunoResearch). Bands were visualized using Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific).
8
Immunoblotting Antibodies for Influenza A Detection
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Anti-phosphorylated-ERK (1:2,000 dilution, CST9106; Cell Signaling Technology, Danvers, MA, USA), anti-total-ERK2 (1:2,000 dilution, GTX113094; GeneTex, Irvine, CA, USA), anti-vimentin (1:1,000 dilution, sc-6260; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5,000 dilution, GTX100118; GeneTex), anti-actin (1:10,000 dilution, MAB1501; Millipore, Burlington, MA, USA), anti-LaminB1 (1:3,000 dilution, ab16048; Abcam, Cambridge, UK), and anti-NP (1:10,000 dilution for western blotting; 1:2,000 dilution for immunofluorescence, produced in our laboratory) antibodies were used in western blotting. Anti-influenza A (1:1,000 dilution, AB1074; Millipore) antibody was used in IHC staining.
9
Liver Protein Analysis by Western Blot
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Protein isolation from liver tissue and Western blot analysis were performed as previously described (Dou et al., 2018 (link)). The following antibodies were used: anti-phosphorylated-AMPKα (Thr172), anti-AMPKα, anti-PPARα, anti-CPT-1, anti-acetyl-CoA-carboxylase (ACC), anti-phosphorylated-ACC, anti-SREBP-1c, anti-fatty acid synthase (FAS), anti-DGAT-2, anti-P53, anti-Bcl2, anti-Bax, anti-TLR4, anti-JNK, anti-phosphorylated-JNK, anti-phosphorylated-P38, anti-P38, anti-phosphorylated-ERK, anti-ERK, anti-cleaved-caspase-3, and anti-GAPDH (Cell Signalling Technology, Danvers, MA, United States).
10
Modulating T-cell Activation and Apoptosis
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Antibodies against human CD3 and CD28 for the stimulation of T cells were purchased from BioXcell (West Lebanon, NH, USA). Human TNFα recombinant protein was obtained from PeproTech EC Ltd. (London, UK). TRIZOL reagent for RNA isolation, MTT powder (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan), and radioimmunoprecipitation assay (RIPA) buffer for cell lysis were provided by Sigma Chemical Co. (St. Louis, MO, USA). Staining reagents for AnnexinV (green) and caspase3/7 (red) were purchased from Essen Bio (Ann Arbor, MI, USA). SYBR Premix Ex Taq was obtained from TaKaRa (Shiga, Japan). Anti-p65, anti-LaminB, anti-IκBα, anti-phosphorylated IκBα, anti-phosphorylated ERK, anti-phosphorylated p38, anti-p38, anti-phosphorylated JNK, and anti-JNK were provided by Cell Signaling Technology (Danvers, MA, USA). Antibodies against actin and ERK were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). ECL Western blotting detection reagents were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The RT PreMix kit was provided by Enzynomics (Daejeon, Korea). DSS was purchased from MP Biomedicals (Irvine, CA, USA).
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