inactive erk2  (Carna Inc)

Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin inhibits MAPK signaling pathway through suppressing CRAF and MEK1/2 but not BRAF kinase activity. a , b The inhibitory effect of erianin on the activity of MEK1 and MEK2 kinase. Active GST-MEK1 full length or GST-MEK2 full length (60 ng) and various doses of erianin were incubated with inactive GST-ERK1 or tag free ERK2 (400 ng) as substrate at 30 °C for 30 min. The phosphorylation of ERK1/2 (Thr202/Tyr204) was detected by western blotting. c The inhibitory effect of erianin on the activity of CRAF kinase. Active CRAF (306-end) (50 ng) and various doses of erianin were incubated with inactive GST-MEK1 (600 ng) as substrate at 30 °C for 30 min. d – f Quantifications of integrated density in ( a – c ) were performed. Data were shown as means ± S.D. of three independent experiments. The asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate a significant difference in the expression of phosphorylation of ERK1 or ERK2 vs total ERK1 or ERK2 in control and erianin-treated group. g The luminescent ADP detection assay was developed to detect the luminescence signal of ATP-to-ADP using the same concentration kinases and substrates described in above kinase assay. Three independent repeats were conducted in this experiment. h Immunoprecipitation (IP)/WB of endogenous CRAF from lysates of SK-MEL-2 (NRAS mut) and A375 (BRAF V600E) cells treated with DMSO or erianin at 12.5, 25, 50 nM for 24 h. Total lysates were immunoblotted for BRAF, CRAF, and MEK1. i IP/WB of endogenous MEK1 from lysates of SK-MEL-2 and A375 cells treated with DMSO or erianin at 12.5, 25, 50 nM for 24 h. Total lysates were immunoblotted for CRAF and MEK1. j , k Western blotting of phospho-CRAF, phospho-MEK1/2 and phospho-ERK1/2 by erianin, vemurafenib, cobimetinib or LY3009120 at indicated concentration for 24 h in NRAS mutant SK-MEL-2 and BRAF V600E mutant A375 cell lines. l Western blotting of MAPK signa l ing pathway by erianin, vemurafenib, cobimetinib, or LY3009120 at indicated concentrations for 24 h in KRAS mutant HCT116 cell line

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: Activity Assay, Incubation, Phospho-proteomics, Western Blot, Expressing, Control, Detection Assay, Concentration Assay, Kinase Assay, Immunoprecipitation, Mutagenesis

Journal: Cancer cell

Article Title: MAST1 drives cisplatin resistance in human cancers by rewiring cRaf independent MEK activation

doi: 10.1016/j.ccell.2018.06.012

Figure Lengend Snippet: (A) MAST1 in vitro kinase assay using MAST1 wild type (WT) or kinase-dead mutant (DA; D497A). GST-MAST1 variants were enriched from 293T and kinase activity was assessed by ADP-Glo Kinase assay using myelin basic protein (MBP) as a substrate. (B) Cell viability of parental cancer cells with WT or DA MAST1 overexpression in the presence of cisplatin. (C) Cell viability of cisplatin-resistant (cisR) cells with endogenous MAST1 knockdown and expression of shRNA-resistant WT or DA MAST1. Relative viability was obtained by normalizing values to cisplatin untreated samples for (B) and (C). (D) Phospho-antibody array results using 1,318 antibodies in KB-3-1cisR lysates. Top seven protein factors whose phosphorylation states decreased in MAST1 knockdown and cisplatin treatment are shown. (E) Parental and cisR pairs of KB-3-1 and A549 cells with MAST1 knockdown and cisplatin treatment (5 μg/ml) were assayed for MEK1 phosphorylation by immunoblotting. (F) MAST1 in vitro kinase assay using recombinant inactive MEK1 (rMEK1 K79A) as a substrate. (G) Kinase activities of MEK1, cRaf and MAST1 in cells with MAST1 knockdown and cisplatin treatment. MEK1, cRaf and MAST1 were immunoprecipitated from KB-3-1 cells and kinase activities were determined by ADP-Glo kinase assay using recombinant inactive ERK2 or MEK1 as substrates. MEK inhibitor U0126 (10 μM) and Raf inhibitor L779450 (5 μM) were used as controls. (H) Western blot analysis of apoptosis-related factors. Cells with or without MAST1 shRNA were treated with cisplatin (5 μg/ml) for 24 hr. (I) Apoptosis assay using parental and cisR cells with or without MAST1 knockdown. Cells were treated with sublethal dose of cisplatin (2 μg/ml: parental, 5 μg/ml: cisR) for 48 hr and apoptotic cells were assayed by Annexin V staining. Error bars represent ± SD from three technical replicates. p values were determined by two-tailed Student’s t test (ns: not significant; *: 0.01 < p < 0.05; **: p < 0.01). See also Figure S2.

Article Snippet: Recombinant inactive ERK2 , SignalChem , Cat#M28–14G.

Techniques: In Vitro, Kinase Assay, Mutagenesis, Activity Assay, Over Expression, Knockdown, Expressing, shRNA, Ab Array, Phospho-proteomics, Western Blot, Recombinant, Immunoprecipitation, Apoptosis Assay, Staining, Two Tailed Test

Journal: Cancer cell

Article Title: MAST1 drives cisplatin resistance in human cancers by rewiring cRaf independent MEK activation

doi: 10.1016/j.ccell.2018.06.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant inactive ERK2 , SignalChem , Cat#M28–14G.

Techniques: Recombinant, Negative Control, Viability Assay, Kinase Assay, Extraction, Ab Array, Activation Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription, shRNA, Sequencing, Plasmid Preparation, Software