Total protein concentration was measured using bradford protein assay (Bio-Rad, Hercules, CA, USA). SDS-PAGE was used for separation of whole protein lysates, following which it was transferred to Polyvinylidene Difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). Blots were incubated with primary antibodies for overnight with gentle shaking at 4 °C. Primary antibodies used were mouse tubulin (Sigma-Aldrich, St. Louis, MO, USA), mouse GRP78 and Gadd 153 (Santa Cruz Biotechnology, Dallas TX, USA)31 (link). The membranes were then incubated with anti-mouse or rabbit secondary antibodies for 1 hour (Jackson Immuno Research Laboratories, West Grove, PA, USA). Florescence was detected using LI-COR Odyssey machine (LI-COR Odyssey CLX, Lincoln, NE, USA).
Gadd153
Manufactured by Santa Cruz Biotechnology
Sourced in United States
GADD153 is a protein that plays a role in the cellular stress response. It is a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors and is involved in the regulation of gene expression during cellular stress conditions. The primary function of GADD153 is to mediate cellular responses to various types of stress, including DNA damage, endoplasmic reticulum stress, and oxidative stress.
Automatically generated - may contain errors
Lab products found in correlation
Grp78, by Santa Cruz Biotechnology (4 mentions)
Bradford protein assay, by Bio-Rad (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Trypsin edta, by Merck Group (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Endog, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ecl western blotting detection kit, by Cytiva (1 mentions)
Beclin 1, by Santa Cruz Biotechnology (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (1 mentions)
Bapta am, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride pmsf, by Merck Group (1 mentions)
Poly adp ribose polymerase parp, by Santa Cruz Biotechnology (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
9 protocols using gadd153
1
Western Blotting Protein Analysis Protocol
2
Immunoblotting Analysis of Cellular Proteins
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Rabbit antibodies against GADD153, GRP78, Beclin-1, and β-actin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), while those against LC-3 were purchased from MBL. Secondary horseradish peroxidase- (HRP-) conjugated goat anti-rabbit and goat anti-mouse antibodies were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Cells were lysed in a buffer containing 10 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), 0.1 mM phenylmethylsulfonyl fluoride, 8 μg/ml aprotinin, and 2 μg/ml leupeptin (pH 7.4). For immunoblotting, proteins resolved by 12.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA), which were then exposed to primary and then secondary antibodies. Chemiluminescence detection was performed with an ECL™ Western Blotting Detection Kit (Amersham). The signal intensities of the corresponding bands were measured by a Light Capture equipped with CS Analyzer software (ATTO, Osaka, Japan).
3
Spectroscopic Identification of Compound K from Panax ginseng
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Compound K (purity > 98%) used in the present study was isolated from P. ginseng, and its structural identities were determined spectroscopically (1H and 13NMR, IR, MS) as previously described [21] (link). RPMI 1640 medium, DMEM medium, fetal bovine serum (FBS), penicillin, streptomycin, and Fura-2/AM were obtained from Life Technologies Inc (Chicago, IL, USA). 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tertazolium bromide (MTT), dimethyl sulfoxide (DMSO), RNase A, phenylmethylsulfonylfluoride (PMSF), propidium iodide (PI), 4-phenylbutyrate (4-PBA), 2-aminoethoxydiphenyl borate (2-APB), dantrolene, BAPTA-AM, ethylene glycol tetraacetic acid (EGTA), and CGP37157 were purchased from Sigma Chemical Co. The following antibodies for caspase-3, caspase-7, caspase-9, poly (ADP-ribose) polymerase (PARP), XBP-1, c-FLIPL, Bid, Bcl-2, Bcl-xL, p-eIF2α, GADD 153, m-calpain, and β-actin were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). The antibodies for X-linked inhibitor of apoptosis protein (XIAP), caspase-8, and caspase-12 were purchased from BD Biosciences, Pharmingen (San Jose, CA, USA). The antibodies for IRE1α and GRP78 were purchased from Cell Signaling Technology (Denvers, MA, USA). z-VAD-fmk, z-ATAD-fmk were purchased from Calbiochem (San Diego, CA, USA).
4
Investigating Cellular Signaling Pathways
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4-Methylumbelliferone was purchased from Sigma-Aldrich (catalog number: M1381; St. Louis, MO, USA). Antibodies against phosphorylated AKT (Ser473, catalog number: 4060), S6 (Ser235/236, catalog number: 2211), ERK1/2 (Thr202/Tyr204, catalog number: 9101), P38 (Thr180/Tyr182, catalog number: 4511), and JNK (Thr183/Tyr185, catalog number: 4668) and for total AKT (catalog number: 9272), S6 (catalog number: 2217), ERK1/2 (catalog number: 4695), P38 (catalog number: 9212), and JNK (catalog number: 9252) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibodies against ATF6α (catalog number: sc-166659), GRP78 (catalog number: sc-13968), GADD153 (catalog number: sc-7351), and α-tubulin (TUBA, catalog number: sc-32293) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The PI3K/AKT signaling pathway inhibitor LY294002 (catalog number: 9901) was purchased from Cell Signaling Technology, Inc. The ERK1/2 inhibitor U-0126 (catalog number: BML-EI282), the P38 inhibitor SB203580 (catalog number: BML-EI286), and the JNK inhibitor SP600125 (catalog number: BML-EI305) were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA).
5
Protein Expression Analysis by Western Blot
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Cell lysis and Western blot analysis were performed as described previously (92 (link)). Briefly, equal amounts of protein (20 μg) were run on SDS–polyacrylamide gel electrophoresis gels, transferred to nitrocellulose membranes, and immunoblotted with the following antibodies: p-eIF2α (1:500; #9721, Cell Signaling Technology, Danvers, MA), p-PERK (1:500; #3191, Cell Signaling), and growth arrest and DNA damage–inducible protein (GADD153)/CHOP (1:500; sc-575, Santa Cruz Biotechnology, Dallas, TX). Following incubation with secondary antibody conjugated to horseradish peroxidase, the respective bands were detected by enhanced chemiluminesence (RPN2106, Amersham Biosciences, Piscataway, NJ). Immunoblots were scanned via Scion Image software.
6
Bufalin-induced Apoptosis Pathway
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Bufalin of 99% purity, dimethyl sulfoxide (DMSO), 4′,6-Diamidino-2-Phenylindole, Dilactate, (DAPI), propidium iodide (PI), and Trypsin-EDTA were obtained from Sigma Chemical Co. (St. Louis, MO, USA). McCoy’s 5A medium, fetal bovine serum (FBS), l -glutamine, and penicillin-streptomycin were purchased from GIBCO®/Invitrogen Life Technologies (Carlsbad, CA, USA). Primary antibody anti-β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA); anti-Bcl-2, Bcl-x, Bax, caspase-3, caspase-8, caspase-9, and PARP were purchased from Cell Signaling Technology (Beverly, MA, USA); anti-cytochrome c, AIF, Endo G, Calpain 1, GRP-78, and GADD153 were purchased from Santa Cruz (Santa Cruz, CA, USA); anti-Fas and caspase-4 were purchased from BD Biosciences (San Diego, CA, USA); and anti-Fas-Ligand was purchased from Millipore (Bedford, MA, USA). Second antibody goat anti-mouse IgG-HRP, goat anti-rabbit IgG-HRP, and goat anti-goat IgG-HRP were purchased from Santa Cruz (Santa Cruz, CA, USA). Second antibody FITC (fluorescein isothiocyanate)-labeled goat anti–mouse IgG Ab, FITC-labeled goat anti-rabbit IgG Ab, and FITC-labeled goat anti-goat IgG Ab were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Bufalin was dissolved in DMSO.
7
Western Blot Analysis of Stress Markers
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Cells were lysed with cell lysis buffer (Cell-Signaling Technology), and the extracted protein fraction was quantified by Bradford protein assay (Bio-Rad laboratories, Hercules, CA, USA) to ensure equal loading of 50 µg of protein for each sample. Samples were loaded into NuPAGE® 4%-12% Bis-Tris Precast Gels (Thermo Scientific, UK) for electrophoresis. Protein bands were transferred from gel onto polyvinylidenedifluoride (PVDF) membranes, and incubated with primary antibodies-Grp78, GADD153 or cleaved caspase-3 p17 (H-60) (rabbit polyclonal antibody; Santa Cruz Biotechnology, UK) at dilution factor of 1:300. On the second day, membranes were incubated with secondary antibody (anti-rabbit HRP conjugated, Cell-Signaling Technology) for 1 h at room temperature at dilution factor of 1:1000, before development with enhanced chemiluminescence (ECL) system (Santa Cruz, Dallas, TX, USA). Protein bands were visualized by Syngene GeneSnap software (Syngene, Cambridge, UK). The intensity of grey scale of protein bands was assessed with ImageJ.
8
Immunofluorescent Staining of Retinal Regions
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Immunofluorescent staining was performed on cryosections. Slides were incubated in 4% paraformaldehyde at room temperature for 30 minutes. After blocking with 10% normal serum for 1 hour, sections were incubated with primary antibodies against 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP), also known as growth arrest- and DNA damage-inducible gene 153 (GADD153) (Santa Cruz, Dallas, TX, USA), at 4°C overnight and 1:100 dilution. The following day, sections were incubated in Alexa Fluor 488–conjugated secondary antibodies (Thermo Fisher, Waltham, MA, USA) for 1.5 hours at 1:250 dilution. Images of the stained sections were acquired using an Olympus BX41 microscope connected to an Olympus DP71 camera. Immunofluorescent staining intensity was quantified and normalized to retinal area using ImageJ (v1.48; National Institutes of Health, Bethesda, MD, USA) software. The first third of the frontal cross sections, taken from the front/corneal side of the eye, corresponds to the peripheral retinal region, the next third corresponds to the midretinal region, and the final third taken nearest to the optic nerve corresponds to the central retinal region.
9
Investigating Cell Death Pathways in Lung Cancer
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Chemicals and reagents. DMC, dimethyl sulfoxide (DMSO), propidium iodide (PI) and Trypsin-EDTA were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Culture medium RPMI-1640, fetal bovine serum (FBS), L-glutamine and penicillin-streptomycin were purchased from Gibco/Invitrogen Life Technologies (Carlsbad, CA, USA). Primary antibodies (anti-AIF, -Endo G, -GRP78, -GADD153, -IRE1β, -ATF-6α, -ATF-6β, and -caspase-4) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
Cell culture. The human lung cancer cell line NCI-H460 was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). The cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine and were incubated in a 5% CO 2 humidified incubator at 37˚C in a 75-cm 2 tissue culture flask.
Cell culture. The human lung cancer cell line NCI-H460 was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). The cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine and were incubated in a 5% CO 2 humidified incubator at 37˚C in a 75-cm 2 tissue culture flask.
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