Journal: Frontiers in Oncology

Article Title: Proteomics and phosphoproteomics of chordoma biopsies reveal alterations in multiple pathways and aberrant kinases activities

doi: 10.3389/fonc.2022.941046

Figure Lengend Snippet: KiPNA analysis and the expression validation of specific proteins. (A) Kinase-pathway network analysis (KiPNA) indicating the proteomic and phosphoproteomic signaling network in chordoma. (B) Fourteen and 29 kinases were conjectured to be negatively and positively regulated, respectively. (C) The elevated expression of brachyury-T, AURA, MOK, and CDK9 were verified by western immunoblotting analysis. The samples were derived from six independent patients who were distinct from the patients whose samples were used for mass spectrometry. T, tumor tissues; N, normal tissues. (D) Densitometric quantification of western blot results from (B) and presented relative to β-actin expression. The data represent the mean ± SD of each experiment carried out in triplicate.

Article Snippet: Briefly, samples were incubated with primary anti-Brachyury antibody (81694, CST, USA, 1:200 dilution) at 37°C for 1 hour and second antibody at 37°C for 30 min. Then, the samples were treated with the DAB Substrate kit (PV-8000, ZSGB-BIO, China).

Techniques: Expressing, Western Blot, Derivative Assay, Mass Spectrometry

Journal: Cell chemical biology

Article Title: Targeted Degradation of Transcription Factors by TRAFTACs: TRAnscription Factor Targeting Chimeras

doi: 10.1016/j.chembiol.2021.03.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cell lines, other reagents and antibodies were purchased from commercial vendors described in the . table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies ECL rabbit IgG, HRP-linked whole Ab GE Health care Cat#NA934; RRID: AB_772206 ECL mouse IgG, HRP-linked whole Ab GE Health care Cat# NA931; RRID: AB_772210 GAPDH (14C10) Cell Signaling Cat# 2118S; RRID: AB_561053 alpha tubulin: Alexa Fluor 488 Invitrogen Cat#53-4502-82;RRID:AB_1210525 NFkB1 p105/p50 Cell Signaling Cat# 3035S; RRID: AB_330564 NFkB p65 Cell Signaling Cat# 8242S; RRID: AB_10859369 brachyury Cell Signaling Cat# 81694S; RRID: AB_2799983 c-Myc (9E10) Santa Cruz Cat# sc-40; RRID: AB_627268 HaloTag Promega Cat# G921A; RRID: AB_2688011 HA-tag Cell Signaling Cat# 3724S; RRID: AB_1549585 DYKDDDDK Tag Cell Signaling Cat# 14793S; RRID: AB_2572291 Bacterial and virus strains E. coli BL21-RIPL codon plus Agilent Technologies Cat# 230280 NEB 5-alpha New England BioLabs Cat# C2987H Rosetta 2 DE3 Sigma Cat# 71397 Chemicals, peptides, and recombinant proteins Tumor Necrosis Factor-α human recombinant protein Sigma Cat# T0157–10UG dCas9 recombinant protein Crews Lab N/A Ni-NTA agarose QIAGEN Cat# 30250 Cycloheximide Sigma Cat# C104450 RNAiMAX transfection reagent Thermo Fisher Sci Cat# 13778–150 MLN4924 selleckchem Cat# S7109 Janelia Fluor 646 (JF646) HaloTag Ligand Promega Cat# ga1120 Critical commercial assays High-Capacity cDNA Reverse Transcription Kit Applied Biosystem Cat# 4368814 KAPA SYBR FAST qPCR Master Mix Kit Kapa Biosystems Cat# KK4600 Experimental models: cell lines HEK293T ATCC Cat# CRL-3216 Flp-In T-Rex 293 Thermo Fisher Sci Cat# {"type":"entrez-nucleotide","attrs":{"text":"R78007","term_id":"853117","term_text":"R78007"}} R78007 HeLa ATCC Cat# CCL-2 Experimental models: organisms/strains Zebrafish ( Danio rerio ) wildtype strain TL Scott Holley Lab, Yale University N/A Oligonucleotides F_CTdCas9:ACCTGACTATGCTGGAGTG GATAAGAAATACUCAATAGGCTTAGCT ATCGGC Yale School of Medicine N/A R_CTdCas9:ATCAGCGGGTTTAACCGGA AATCUCCAGAGTAGACAGCC Yale School of Medicine N/A F_CTPCDNA5:AGATTTCCGGTTAAACC CGCTGAUCAGCCTCGAC Yale School of Medicine N/A R_CTPCDNA5:AGTATTTCTTATCCACTC CAGCATAGTCAGGUACGTCATAAGGG Yale School of Medicine N/A RNA:DNA chimeric oligos This manuscript Figure S1 pCDNA5-HA-NTdCas9HT7 This manuscript Table S1 pCDNA5-HA-NTdCas9HT7-NLS This manuscript Table S1 Recombinant DNA pET302-6His-dCas9-Halo Deng et al Proc Natl Acad.

Techniques: Recombinant, Transfection, Expressing, Software