Phospho p44 42 mapk erk1 2 thr202 tyr204 d13.14.4e

Manufactured by Cell Signaling Technology

Sourced in China, Italy

Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) is a lab equipment product that detects the phosphorylation of p44/42 MAPK (Erk1/2) at Thr202/Tyr204 residues. It is a primary antibody used in Western blotting and immunohistochemistry applications.

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7 protocols using phospho p44 42 mapk erk1 2 thr202 tyr204 d13.14.4e

Antibody-Based Protein Expression Analysis

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The following mouse monoclonal antibodies were used: v-Src (#327, Calbiochem), Src (GD11, Millipore), phosphotyrosine (pTyr, 4G10, Upstate Biotechnology), and actin (C4, Millipore). The following rabbit monoclonal antibodies were used: phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E, Cell Signaling Technology), and p21 Waf1/Cip1 (12D1, Cell Signaling Technology). The following rabbit polyclonal antibodies were used: c-Src (N-16, Santa Cruz Biotechnology), ERK2 (C-14, Santa Cruz Biotechnology), Src[pY⁴¹⁶] (phospho-Src family, Cell Signaling Technology), p16 (C-20, Santa Cruz Biotechnology), p21 (C-19, Santa Cruz Biotechnology) and Cleaved Caspase-3 (Asp175, Cell Signaling Technology). The following rat monoclonal antibody was used: α-tubulin (MCA78G, Serotec). Horseradish peroxidase-F(ab’)₂ fragments of anti-mouse IgG antibody (GE Healthcare), anti-rabbit IgG antibody (Cell LAB), and anti-rat IgG antibody (GE Healthcare) were used. Alexa Fluor 488-donkey-anti-mouse IgG and Alexa Fluor 647-goat-anti-mouse IgG secondary antibodies were obtained from Invitrogen.

Honda T., Morii M., Nakayama Y., Suzuki K., Yamaguchi N, & Yamaguchi N. (2018). v-Src-driven transformation is due to chromosome abnormalities but not Src-mediated growth signaling. Scientific Reports, 8, 1063.

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Immunofluorescence Staining of Phospho-ERK1/2

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Cells were seeded on a 12-well plate to reach 50% confluence. All the following procedures were performed at RT unless otherwise noted. Cells were at first fixed in 4% paraformaldehyde (PFA) for 30 min and permeabilized in 0.5% Triton X-100 for 20 min. Then, cells were incubated for 1 h in the blocking solution (Beyotime, China). Afterwards, cells were stained with Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) (#4730S, 1:200, Cell Signaling Technology) at + 4 °C overnight, followed by staining with anti-rabbit Alexa594 (1:500, Cell Signaling Technology) secondary antibody for 1 h. After three times washes in PBS for 10 min, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (KeyGen BioTECH, China) for 5 min. Eventually, cells were imaged using a fluorescent microscope (Olympus, Tokyo, Japan).

Mai M., Guo X., Huang Y., Zhang W., Xu Y., Zhang Y., Bai X., Wu J, & Zu H. (2022). DHCR24 Knockdown Induces Tau Hyperphosphorylation at Thr181, Ser199, Ser262, and Ser396 Sites via Activation of the Lipid Raft-Dependent Ras/MEK/ERK Signaling Pathway in C8D1A Astrocytes. Molecular Neurobiology, 59(9), 5856-5873.

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Autophagy Modulation by ULK1/2 Inhibitors

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IFNγ recombinant human (# PHC4033) and mouse (# PMC4033) proteins were from Gibco (Life Technologies). MRT68921 dual autophagy kinase ULK1/2 inhibitor was purchased from Selleckchem (# S7949). Chloroquine (CQ) was purchased from Sigma-Aldrich. XMD8–92 was purchased from Tocris (# 4132). The Edit-R human lentiviral ULK1 sgRNAs (# VSGH10142–246477203) and Edit-R Lentiviral hEF1α-Blast-Cas9 nuclease plasmid DNA (# CAS10138) were purchased from GE Healthcare Dharmacon. ON-TARGETplus Non-targeting Control Pool siRNA (# D-001810–10-05), ON-TARGETplus PIK3C3 siRNA (SMARTpool: # L-005250–00-0005) and ON-TARGETplus BECN1 siRNA (SMARTpool: # L-010552–00-0005) were purchased from Dharmacon. Antibodies against ULK1 (D8H5) (#8054), MLK3 (#2817), phospho-ERK5 (Thr²¹⁸/Tyr²²⁰) (#3371), ERK5 (D3I5V) (#12950), phospho-p44/42 MAPK (ERK1/2) (Thr²⁰²/Tyr²⁰⁴) (D13.14.4E) (#4370), p44/42 MAPK (ERK1/2) (#9102), phospho-SAPK/JNK (Thr¹⁸³/Tyr¹⁸⁵) (81E11) (#4668), SAPK/JNK (#9252), phospho-STAT1 (Ser⁷²⁷) (D3B7) (#8826), phospho-STAT1 (Tyr⁷⁰¹) (D4A7) (#7649), phospho-p90RSK (Thr³⁵⁹/Ser³⁶³) antibody (#9344), and RSK1 (D6D5) (#8408) were purchased from Cell Signaling. Anti-MLK3 (phospho Thr²⁷⁷ + Ser²⁸¹) antibody (#ab191530) was purchased from abcam and STAT1 p84/p91 antibody (E-23) (#sc-346) was from Santa Cruz Biotechnology. Antibody against GAPDH (6C5) (#MAB374) was purchased from EMD Millipore.

Saleiro D., Blyth G.T., Kosciuczuk E.M., Ozark P.A., Majchrzak-Kita B., Arslan A.D., Fischietti M., Reddy N.K., Horvath C.M., Davis R.J., Fish E.N, & Platanias L.C. (2018). IFNγ-inducible antiviral responses require ULK1-mediated activation of MLK3 and ERK5. Science signaling, 11(557), eaap9921.

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Western Blot Analysis of Protein Signaling

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Cells and xenograft tumor samples were resuspended in high SDS-RIPA Buffer (50 mM Tris-HCl, pH 7.5, 150 mM Sodium Chloride, 1 % Triton X-100, 1 % sodium deoxycholate, 1 % SDS, 2 mM EDTA; Sigma Aldrich). Tissues were disrupted and homogenized with a TissueLyser II (Qiagen) for 2 × 2 min intervals at 30Hz. Protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Pierce). A total of 15–50 μg of protein extracts were loaded onto NuPAGE® Novex® 4–12 % Bis-Tris Protein Gels (Life Technologies) and subsequently transferred onto nitrocellulose membranes using the iBlot® Dry Blotting System (Life Technologies). The blots were developed using SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific). Antibodies: S6-Ribosomal protein (5G10), Phospho-S6 Ribosomal protein (Ser240/244) (D68F8), Phospho-4E-BP1 (Thr37/46) (236B4), p44/42 MAPK (Erk1/2) (137 F5), and Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) were purchased from Cell Signaling Technology. C-MYC (Y69) and N-MYC (NCM II 100) were purchased from Abcam. FLAG (M2) and β-actin (A2066) antibodies were purchased from Sigma Aldrich.

Dela Cruz F.S., Diolaiti D., Turk A.T., Rainey A.R., Ambesi-Impiombato A., Andrews S.J., Mansukhani M.M., Nagy P.L., Alvarez M.J., Califano A., Forouhar F., Modzelewski B., Mitchell C.M., Yamashiro D.J., Marks L.J., Bender J.L, & Kung A.L. (2016). A case study of an integrative genomic and experimental therapeutic approach for rare tumors: identification of vulnerabilities in a pediatric poorly differentiated carcinoma. Genome Medicine, 8, 116.

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Western Blot Analysis of Fibroblast Proteins

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Fibroblasts were collected and suspended in RIPA buffer (Radio-Immuno Precipitation Assay Buffer). Forty micrograms of total protein were analyzed by 10% denaturing polyacrylamide gels and then transferred electrophoretically to PVDF membranes (Immobilon-NC, Millipore, Italy). Anti-A2BR (H-40) (Santa Cruz Biotechnology), anti HIF1α (A300-286A) (Bethyl Laboratories, Tema Ricerca, Italy) or anti CD73 (5NT5E, C-terminal) (Sigma-Aldrich, Milan, Italy) or phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) or p44/42 MAPK (Erk1/2) (137F5) (Cell Signaling Technology) primary antibodies were used. Immunoreactive protein bands were visualized by enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK) and analyzed to Las4000 (GE Healthcare Life Sciences).

Sorrentino C., Miele L., Porta A., Pinto A, & Morello S. (2016). Activation of the A2B adenosine receptor in B16 melanomas induces CXCL12 expression in FAP-positive tumor stromal cells, enhancing tumor progression. Oncotarget, 7(39), 64274-64288.

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Cellular Signaling Pathway Analysis

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A549 and H2228 cells were plated in six-well plates at a confluency of 70%. 48 hours after adenovirus infection, whole-cell extracts were prepared by lysing cells with the addition of 500 µL of hot SDS-PAGE buffer (Beyotime, P0015B). Tumor tissues were homogenized by TGrinder (Tiangen, OSE-Y30), and lysed with RIPA buffer containing complete protease inhibitor cocktail (Roche, 11697498001). Target proteins were detected by western blot analysis with the following antibodies: GAPDH mouse monoclonal antibody (Proteintech, 60004-1-Ig,), Akt (pan) (40D4) mouse monoclonal antibody (Cell Signaling Technology, 2920), Phospho-Akt (Ser473) (D9E) XP Rabbit mAb (Cell Signaling Technology, 4060), p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb (Cell Signaling Technology, 4695), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) (Cell Signaling Technology, 4370), mouse monoclonal Anti-MAP Kinase, activated (Diphosphorylated ERK-1&2) antibody (Sigma-Aldrich, M8159), Ras Antibody (Cell Signaling Technology, 3965), and Anti-RAS (G12S) Mouse Monoclonal Antibody (NewEast Biosciences, 26186).

Gao Q., Ouyang W., Kang B., Han X., Xiong Y., Ding R., Li Y., Wang F., Huang L., Chen L., Wang D., Dong X., Zhang Z., Li Y., Ze B., Hou Y., Yang H., Ma Y., Gu Y, & Chao C.C. (2020). Selective targeting of the oncogenic KRAS G12S mutant allele by CRISPR/Cas9 induces efficient tumor regression. Theranostics, 10(11), 5137-5153.

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Quantitative MAPK Activation Assay

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Proteins from suspension cells and plants were extracted as described (Holley et al., 2003 (link)). Thirty to 50 μg total protein was analyzed for MAPK phosphorylation by immunoblotting using an anti-phospho-ERK antibody (phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204); D13.14.4E; Cell Signaling Technology) at a dilution of 1:2,500 (1:2,000 for grasses) in 5% BSA as described (Dombrowski et al., 2011 (link)). This antibody specifically recognizes active MAPKs that are dually phosphorylated on a threonine and tyrosine residue in a TEY phosphorylation motif, which is conserved among certain plant and metazoan MAPKs (Hind et al., 2010 (link); Bethke et al., 2012 (link)). In grasses, MAPKs were detected using an alkaline phosphatase NBT/BCIP system as described (Dombrowski et al., 2011 (link)). In dicots, MAPKs were detected by X-ray film using a chemiluminescence assay (Lumi-Phos substrate, Thermo Scientific, Fisher Scientific) with a anti-rabbit IgG secondary antibody coupled to alkaline phosphatase (Sigma–Aldrich). Equal protein loading was confirmed by staining proteins on membranes with coomassie brilliant blue (CBB). MAPK signals on immunoblots were quantified using ImageQuant TL software (GE Healthcare).

Hann C.T., Bequette C.J., Dombrowski J.E, & Stratmann J.W. (2014). Methanol and ethanol modulate responses to danger- and microbe-associated molecular patterns. Frontiers in Plant Science, 5, 550.

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