P atf2

Manufactured by Cell Signaling Technology

Sourced in United States

P-ATF2 is an antibody that recognizes the phosphorylated form of the Activating Transcription Factor 2 (ATF2) protein. ATF2 is a transcription factor that plays a role in cellular stress response pathways.

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Anti-p-ATF2 (T71)

18 protocols using p atf2

Protein Extraction and Western Blot Analysis

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Cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.1% SDS, 0.5% (w/v) sodium deoxycholate and protease inhibitors: 1 mM PMSF and complete inhibitor cocktail [Roche]) on ice for 30 min. Lysates were centrifuged at 20,000 g for 15 min and the supernatant was collected. Protein content was measured by the DC protein assay (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Equal amounts of protein from each sample were resolved on 12% Bis-Tris pre-cast gels (Invitrogen) and transferred to a PVDF membrane (Millipore, Bellerica, MA, USA). Membranes were blocked with 5% low-fat milk in PBS supplemented with 0.1% Tween 20 (PBST) and then incubated with various primary antibodies followed by a secondary horseradish peroxidase-conjugated antibody. Membranes were washed after the primary and secondary antibodies three times in PBST.
The antibody for Livin was purchased from Imgenex (San Diego, CA, USA). Antibodies for caspase 3, caspase 9, P-JNK, JNK, P-ATF2, ATF2, c-JUN, P-p38, p38 and β-Actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies for P-c-JUN, JIP-1 and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody for PARP was purchased from Enzo Life Sciences.

Shiloach T., Berens C., Danke C., Waiskopf O., Perlman R, & Ben-Yehuda D. (2014). tLivin Displays Flexibility by Promoting Alternative Cell Death Mechanisms. PLoS ONE, 9(6), e101075.

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Western Blot Analysis of Cell Signaling Pathways

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Total protein
extracts were generated using lysis buffer (50 mM Tris-HCl, pH 7.4,
150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1
mM EDTA) supplemented with PhosSTOP and Complete Phosphatase/Protease
Inhibitor Cocktails (Roche Diagnostics GmbH, Mannheim, Germany). Protein
extracts (20–25 μg per sample) were loaded onto sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels
and transferred electrophoretically to poly(vinylidene fluoride) (PVDF)
membranes. The following primary antibodies were used at a dilution
of 1:1000: ERK1/2 (p44/42) (ab17942, Abcam), p-ERK1/2 (p-p44/42) (Thr202/Tyr204)
(9101, Cell Signaling), MNK1 (2195, Cell Signaling), p-MNK1 (Thr197/202)
(2111, Cell Signaling), eIF4E (9742, Cell Signaling), p-eIF4E (p-Ser
209) (9741, Cell Signaling or NBP2-66802, Novus Biologicals), p38alpha
(9218, Cell Signaling), p38beta (2339, Cell Signaling), p-p38 (4511,
Cell Signaling), p-ATF2 (27934, Cell Signaling), Hsp27 (2402, Cell
Signaling), p-Hsp27 (S82) (2401, Cell Signaling), and p-p90 RSK (Thr573)
(9346, Cell Signaling). The primary HRP-conjugated antibody anti-β-actin
(Calbiochem) was used at a dilution of 1:20 000. Anti-mouse
and anti-rabbit HRP secondary antibodies were from Pierce and used
at a dilution of 1:10 000. Immunodetection of proteins was
performed using ECL Western Blotting Detection Reagents (GE Healthcare,
Buckinghamshire, U.K.).

Bou-Petit E., Hümmer S., Alarcon H., Slobodnyuk K., Cano-Galietero M., Fuentes P., Guijarro P.J., Muñoz M.J., Suarez-Cabrera L., Santamaria A., Estrada-Tejedor R., Borrell J.I, & Ramón y Cajal S. (2022). Overcoming Paradoxical Kinase Priming by a Novel MNK1 Inhibitor. Journal of Medicinal Chemistry, 65(8), 6070-6087.

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Plasmid and Antibody Protocol for MCPyV Signaling

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pcDNA6 MCV STco (codon optimised) was a gift from Patrick Moore (Addgene plasmid #40201; http://n2t.net/addgene:40201; RRID:Addgene_40201). pcDNA3.1-Flag-PP4C and RL-PP4C-TDN-HA were gifts from Marilyn Goudrealt (University of Toronto, Canada) and Tse-Hua Tan (National Health Research Institute, Taiwan), respectively. Primary antibodies targeting P-ERK1/2, ERK1/2 total, P-p38, p38 total, P-MK2, MK2 total, P-MSK1, MSK1 total, P-ATF2, ATF2 total, P-MKK4, MKK4 total and P-MKK3/6 were purchased from Cell Signalling Technologies and diluted 1 : 1000 in tris buffered saline (pH 7.6), containing 1% tween 20 (TBS-T) and 5% BSA, with the exception of P-ERK1/2 which was diluted 1 : 2000. GAPDH antibodies were purchased from Abcam and diluted 1 : 10 000 in TBS-T containing 5% non-fat milk. MCPyV ST was detected using 2T2 antibodies, kindly provided by Christopher Buck (NCI/CCR, Maryland, U.S.A.). Secondary antibodies conjugated to HRP were purchased from Dako (anti-mouse) and Cell Signaling Technologies (anti-rabbit) and diluted in TBS-T containing 5% non-fat milk 1 : 5000 and 1 : 2000, respectively. The p38 inhibitor SB202190 was purchased from Caltag Medsystems and used at 10 μM. SB202474 and U0126 were purchased from Cambridge Bioscience and used at 10 μM and 20 μM, respectively. All drugs were made up in DMSO at 1000× the concentration stated above.

Dobson S.J., Anene A., Boyne J.R., Mankouri J., Macdonald A, & Whitehouse A. (2020). Merkel cell polyomavirus small tumour antigen activates the p38 MAPK pathway to enhance cellular motility. Biochemical Journal, 477(14), 2721-2733.

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Investigating Transcription Factor Activation

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LECs (2×10⁶) treated with Stattic, SP600125, IL6 or EGM, were used to prepare cell lysates or nuclear extracts. Five hundred microliters of cell lysates or 200 µL nuclear extracts were incubated overnight at 4°C with antibodies suitable for IP (1:100 diluted): pStat3, pc-Jun, pATF-2, pNFkB, and NFkB (Cell Signaling). Ten microliters of Protein A/G Plus Agarose (Santa Cruz Biotech) was added and incubated for 3 h at 4°C. The beads were rinsed 3 times with 500 µL cell lysis buffer for IP (Pierce) supplemented with the protease inhibitor and phosphatase inhibitor cocktail 2/3 (Sigma). The protein complex was reduced and separated by SDS-PAGE and probed with the following antibodies in a Western assay: pStat3, pc-Jun, pATF-2, pNFkB, and NFkB (Sigma). All the original gel images of immunoblot analyses are presented in Supplementary Fig. 25.

Lee E., Fertig E.J., Jin K., Sukumar S., Pandey N.B, & Popel A.S. (2014). Breast cancer cells condition lymphatic endothelial cells within pre-metastatic niches to promote metastasis. Nature communications, 5, 4715.

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Western Blot Analysis of Apoptosis Markers

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Rabbit anti-human Bax, ERK, JNK and p-JNK antibodies were obtained from ProteinTech Group (Chicago, IL, USA). Rabbit anti-human antibodies against p-PI3K, AKT, p-AKT, PARP, pro-caspase-3, cleaved-caspase-3, pro-caspase-9, cleaved-caspase-9, Bcl-xl, Bcl-2, Mcl-1, Bad, p38, p-p38, p-ERK, p-ATF2 and p53 were obtained from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-human antibodies against p-Bad and cytochrome C were obtained from Epitomics (Burlingame, CA, USA). Rabbit anti-human β-actin antibodies were obtained from Sigma (St Louis, MO, USA). Goat anti-rabbit and horseradish peroxidase conjugated IgG was obtained from Millipore (Bellerica, MA, USA). Immunoblotting was performed as previously described [19 (link)]. The treated samples and the control samples (25 μg) were separated by 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins on the gel were then transferred to PVDF membranes. The PVDF membranes were incubated with the primary antibody (1:1000 dilutions in 2% dehydrated skim milk) at 4 °C overnight. Then, incubation at 4 °C was performed for 2 h with the secondary antibodies (goat anti-rabbit or goat anti-mouse and horseradish peroxidase conjugate, 1:5000 dilution in 2% dehydrated skim milk). The blots were detected through chemiluminescence using enhanced ECL western blotting kit.

Su C.C., Chen J.Y., Din Z.H., Su J.H., Yang Z.Y., Chen Y.J., Wang R.Y, & Wu Y.J. (2014). 13-Acetoxysarcocrassolide Induces Apoptosis on Human Gastric Carcinoma Cells Through Mitochondria-Related Apoptotic Pathways: p38/JNK Activation and PI3K/AKT Suppression. Marine Drugs, 12(10), 5295-5315.

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Western Blot Analysis of Stress Signaling

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Prepared, immunoprecipitated (IP) samples and corresponding whole cell lysates were resolved by SDS-PAGE and transferred to a nitrocellulose membrane according to the manufacturer’s (BioRad) instructions. Membranes were blocked with 5% BSA in TBS for 30 min, and incubated with primary antibodies against JNK1/2 (1:1000)(Invitrogen), P-SAPK/JNK (1:1000) (Cell Signaling), P-c-jun (1:1000) (Cell Signaling), P-ATF2(1:1000) (Cell Signaling), PRDX1 (1:4000)(Abcam), PRDX-SO3 (1:1000)(Abcam), and actin (1:1000) (Oncogene), overnight at 4 °C. Membranes were washed four times for 5 min each in TBST (0.05% Tween-20) and visualized by IR or chemiluminescent detection. For IR processing, membranes were incubated with a 1:15000 dilution of anti-goat, anti-rabbit, or anti-mouse IRDye (LI-COR), for 30 min at 25 °C. Blots were washed three times with TBST, once with TBS, and imaged on an Odyssey (LI-COR) imager. Membranes processed by chemiluminescence were incubated in a 1:10000 dilution of HRP-conjugated anti-mouse or anti-rabbit antibodies for 1 h at 25 °C. Blots were washed four times with TBST for 5 min and exposed to ECL for 1 min.

Jezierska-Drutel A., Attaran S., Hopkins B.L., Skoko J.J., Rosenzweig S.A, & Neumann C.A. (2019). The peroxidase PRDX1 inhibits the activated phenotype in mammary fibroblasts through regulating c-Jun N-terminal kinases. BMC Cancer, 19, 812.

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Cell Lysis and Protein Analysis

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Proteins from cell were lysed in RIPA buffer (pH = 7.4, 50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholic acid, 1 mM Na3 VO4, 1 M EDTA, 1 mM NaF) containing protease inhibitors cocktail (Amresco, Solon, OH, USA). The protein contents of samples were assayed using a Bio-Rad DC protein assay kit II (Bio-Rad, Hercules, CA, USA). Protein samples were separated by electrophoresis on SDS-PAGE 8%, 10% gels. After blocking in 5% non-fat milk, the membrane was incubated with primary antibodies for pro-PARP, pro-caspase-3, Bax, CHOP, p-eIF2α, ATF4, p-ATF2, PERK, and β-actin (Cell Signaling, Beverly, MA, USA) followed by the exposure to horseradish peroxidase (HRP)-conjugated secondary anti-mouse or rabbit antibodies (Bioss Antibodies, Woburn, MA, USA). To visualize protein bands, a chemiluminescence (ECL) system (Amersham Pharmacia, Piscataway, NJ, USA) was used.

Lim H.J., Park M.N., Kim C., Kang B., Song H.S., Lee H., Kim S.H., Shim B.S, & Kim B. (2019). MiR-657/ATF2 Signaling Pathway Has a Critical Role in Spatholobus suberectus Dunn Extract-Induced Apoptosis in U266 and U937 Cells. Cancers, 11(2), 150.

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Molecular Mechanisms of Herpes Virus Regulation

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Wogonin and acyclovir were obtained from the National Institutes for Food and Drug Control in China (Beijing, China). SB203580, SP600125, and MG132 were purchased from Beyotime Biotechnology Institute (Haimen, Jiangsu, China). Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L), DAPI, DRAQ5 and SYBR green real-time PCR reagent were obtained from Life Technologies, Thermo Fisher Scientific (Carlsbad, CA, USA). IRDye 680-conjugated goat-anti-rabbit and IRDye 800-conjugated goat-anti-mouse antibodies were obtained from LI-COR (Lincoln, NE, USA). Antibodies specific for HSV-1/2 gD, HSV-1 ICP0, HSV-1 ICP4, HSV-1 ICP27, JNK2, p38, GAPDH, and RIPA lysis buffer were purchased from Santa Cruz (Santa Cruz, CA, USA). p65, p-p38, p-c-Jun, p-JNK1/2, p-ATF-2, and IκB-α antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Bright-Glo luciferase assay system was purchased from Promega (Madison, WI, USA).
Vero and HEC-1-A cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). NF-κB-luc and AP-1-luc reporter plasmids were purchased from Clontech (Palo Alto, CA, USA). HSV-1(HF), HSV-1/blue and HSV-2 (G) were propagated and titrated in Vero cells as described previously [36 (link)].

Chu Y., Lv X., Zhang L., Fu X., Song S., Su A., Chen D., Xu L., Wang Y., Wu Z, & Yun Z. (2020). Wogonin inhibits in vitro herpes simplex virus type 1 and 2 infection by modulating cellular NF-κB and MAPK pathways. BMC Microbiology, 20, 227.

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Western Blot of Apoptosis and UPR Markers

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Samples were separated on 4–12% Bis-Tris gels (Invitrogen, Carlsbad, CA). For cytoplasmic fractions or whole cell lysates, 100 μg of total protein was loaded. For nuclear fractions, 10 μg of total protein was loaded. The following antibodies and conditions were used: cleaved PARP (9544; 1:500; Cell Signaling, Danvers, MA), caspase-6 (9762; 1:500; Cell Signaling), cleaved caspase-3 (9664; 1:500; Cell Signaling), XBP-1 (sc-7160; 1:200; Santa Cruz, Dallas, TX), ATF4 (sc-200; 1:200; Santa Cruz), ATF6 (IMG-273; 1:200; Imgenex, San Diego, CA); LRH-1 (PP-H2325-10; 1:200; Perseus Proteomics, Tokyo, Japan), pATF2 (9225S; 1:500; Cell Signaling [note: we have experienced difficulties with recent batches]), ATF2 (ab47476; 1:1000; Abcam, Cambridge, England); and PLK3 (4896S; 1:400; Cell Signaling). Immobilon Western substrate (Millipore, Billerica, MA) was used for detection.

Mamrosh J.L., Lee J.M., Wagner M., Stambrook P.J., Whitby R.J., Sifers R.N., Wu S.P., Tsai M.J., DeMayo F.J, & Moore D.D. (2014). Nuclear receptor LRH-1/NR5A2 is required and targetable for liver endoplasmic reticulum stress resolution. eLife, 3, e01694.

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Investigating Transcription Factor Activation

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