primary antibodies against kras  (Thermo Fisher)

Journal: Nature Communications

Article Title: AIMP2-DX2 provides therapeutic interface to control KRAS-driven tumorigenesis

doi: 10.1038/s41467-022-30149-2

Figure Lengend Snippet: a DX2 levels in H460 lung cancer cells were controlled by the introduction of Strep-DX2 and si-DX2. Protein and mRNA levels were determined by Western blotting (WB) and RT-PCR (RT), respectively. Phosphorylation of ERK and Akt was monitored for KRAS signaling. Actin was used as a loading control. Results are representative of at least three independent experiments. b KRAS level in doxycycline (Dox)-inducible DX2 transgenic mice (Ind-DX2). Levels of KRAS and DX2 in colorectal tissues from two controls (#1, 2) and two Dox-fed mice (#3, 4) were analyzed. c MEF isolated from Ind-DX2 were treated with Dox in a time-dependent manner and protein levels were determined. Results are representative of at least three independent experiments. d Heat map from the results showing DX2-dependent regulation of RAS isoforms. DX2-mediated changes in the levels, stability, and ubiquitination of isoforms were quantified from Supplementary Fig. . Cell viability and anchorage-independent colony formation assay were quantified from Supplementary Fig. . Maximum (max) and minimum (min) values from the indicated experiments were designated as different colors with the highest and lowest intensity, respectively, and the rests were graded according to their relative values. e Result of xenograft ( n = 4) using H460 cells stably expressing the indicated combination of KRAS4B and sh-DX2. Representative images of mice and tumors for each group (left) and tumor sizes (right) were shown. Sh means short hairpin RNA. All error bars represent the standard deviation (S.D.). P value is from the two-sided t test. f Heat map representing the cellular levels of DX2 and KRAS in 18 lung, 12 colorectal and 8 pancreatic cell lines. The cellular levels of DX2 and KRAS in the tested cell lines were represented as the heat map of orange and blue colors, respectively. The maximum and minimum values quantitated as described in Methods were shown with the highest and lowest color intensity, respectively, and the rests were graded according to their relative values. g Analysis of the levels of DX2 and KRAS in lung and colorectal tumor tissues. Staining intensities of the two proteins were classified as low (L) and high (H) (Supplementary Fig. ). Number of the analyzed tissue samples is shown in brackets. h DX2 and KRAS levels in the tumor and matched normal (MN) tissues from 99 patients with colorectal cancer. Depending on the levels in tumor compared to MN, samples were classified as H and L as compared to MN levels (Supplementary Fig. ). Source data are provided as a .

Article Snippet: Primary antibodies against KRAS and DX2 were purchased from Novus Biologicals and CureBio, respectively.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Phospho-proteomics, Control, Transgenic Assay, Isolation, Ubiquitin Proteomics, Colony Assay, Stable Transfection, Expressing, shRNA, Standard Deviation, Staining

Journal: Nature Communications

Article Title: AIMP2-DX2 provides therapeutic interface to control KRAS-driven tumorigenesis

doi: 10.1038/s41467-022-30149-2

Figure Lengend Snippet: a Top 200 proteins identified as the potential binding proteins of DX2 by LC-MS analysis. The relative fold change values of 12 proteins in proliferative ontology were represented as the bar graph (left upper box). Among 12 identified proteins, PRDX1 was detected at the highest fold change and the fold changes of other proteins were divided by that of PRDX1. The relative values of the other eleven proteins were graded, taking the value of PRDX1 as 1. Diameter of circles denotes the relative fold changes of a total of 200 identified proteins. b , c Determination of specific binding of DX2 to KRAS. DX2 was mixed with different nanoluciferase-RAS isoforms. Amounts of the indicated RAS isoforms co-precipitated with DX2 were determined by luciferase activity and are shown as a bar graph ( b ). CCD18CO cells expressing Strep-tagged DX2 and the indicated nanoluciferase-RAS isoforms were precipitated using a strep-tactin column. Amounts of the indicated nanoluciferase-RAS isoforms co-precipitated with DX2 were measured and shown as above ( c ). All the experiments were independently repeated thrice and error bars denote S.D. Data are presented as mean values ± S.D. P value is from the two-sided t test. d In vitro pull-down assay showing direct binding between DX2 and KRAS4B. Co-precipitated KRAS4B or HRAS with GST-DX2 was detected by SDS-PAGE and immunoblotting using an anti-pan RAS antibody. GST proteins were detected by Coomassie staining. Results are representative of at least three independent experiments. e Endogenous KRAS in DLD-1 cells treated with EGF for 30 min was precipitated with the anti-KRAS antibody. p-EGFR was used as a positive control for EGF signaling. Results are representative of at least three independent experiments. f Schematic diagram presenting the binding region between DX2 and KRAS4B. Binding region is depicted as a dashed line (Supplementary Fig. ). g In vitro pull-down assay showing direct interaction between DX2 protein and KRAS4B, but not with HRAS, HVR peptide. Biotin-conjugated and native peptide were serially mixed with DX2 proteins for checking the binding specificity. Results are representative of at least three independent experiments. EV, PD, IP, and WCL represent empty vector, pull-down, immunoprecipitation, and whole cell lysate, respectively. Source data are provided as a .

Article Snippet: Primary antibodies against KRAS and DX2 were purchased from Novus Biologicals and CureBio, respectively.

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Luciferase, Activity Assay, Expressing, In Vitro, Pull Down Assay, SDS Page, Western Blot, Staining, Positive Control, Plasmid Preparation, Immunoprecipitation

Journal: Nature Communications

Article Title: AIMP2-DX2 provides therapeutic interface to control KRAS-driven tumorigenesis

doi: 10.1038/s41467-022-30149-2

Figure Lengend Snippet: a , b Chemical shift perturbation of 15 N-labeled DX2 51-251 (C136S, C222S) by the binding to KRAS4B HVR peptide. Superposition of the 2-dimensional (2D) 1 H- 15 N TROSY spectra of free DX2 in the presence (red) and absence (black) of HVR peptide. Strongly affected residues are depicted ( a , left) and the indicated signals are enlarged and presented ( a , right). The residues showing strong (Δδ > 0.02 ppm) and medium (0.01 < CSP ≤ 0.02 ppm) perturbation are shown in red and orange, respectively, on the surface of the DX2 GST domain ( b ). c Result of 600 ns molecular dynamics (MD) simulation to determine the binding surface of DX2 and KRAS4B. The two significant interfaces are highlighted by the dashed box. d Significant residues for binding between DX2 and KRAS4B were analyzed by immunoprecipitation using alanine mutants (Supplementary Fig. ) and presented in the enlarged image of each interface. e , f Validation of the effect of KRAS4B farnesylation on its binding with DX2. H460 cells expressing Strep-DX2 were treated FTase inhibitor I and separated into cytosol and membrane fraction. HSP90 and cadherin (Cad) were used as loading markers for cytosol (Cyto) and membrane (Memb), respectively ( e ). In vitro pull-down assay showing the binding of DX2 to farnesylated KRAS4B. In vitro farnestlyated KRAS4B proteins were mixed with the purified GST-DX2. Binding of the two proteins and farnesylation were determined by immunoblotting and fluorescent scans, respectively. N and F indicate native and farnesylation, respectively ( f ). Results are representative of at least three independent experiments. g H460 cells introducing Strep-DX2 were treated with EGF and separated into Cyto and Memb fractions. Relative levels of KRAS were presented below the KRAS blots. Results are representative of at least three independent experiments. h Schematic presentation for binding between DX2 and KRAS. un KRAS and ST KRAS mean unstable and stable KRAS, respectively. Source data are provided as a .

Article Snippet: Primary antibodies against KRAS and DX2 were purchased from Novus Biologicals and CureBio, respectively.

Techniques: Labeling, Binding Assay, Immunoprecipitation, Biomarker Discovery, Expressing, Membrane, In Vitro, Pull Down Assay, Purification, Western Blot

Journal: Nature Communications

Article Title: AIMP2-DX2 provides therapeutic interface to control KRAS-driven tumorigenesis

doi: 10.1038/s41467-022-30149-2

Figure Lengend Snippet: a Smurf2-mediated levels of KRAS. Endogenous levels of KRAS from H460 cells expressing Smurf2 wild type (WT), catalytic-inactive mutant (CA), and si-Smurf2 were determined by immunoblotting. si means siRNA. b Smurf2-mediated ubiquitination of KRAS. The cells described in ( a ) were treated with MG-132 and the ubiquitinated amounts of KRAS were analyzed by ubiquitination assay using the anti-ubiquitin (Ub) antibody. c In vitro ubiquitination assay showing the effect of DX2 on Smurf2-mediated ubiquitination of KRAS4B. UBE1, UbcH5c/UBE2D3, Smurf2, and KRAS4B were used as E1, E2, E3, and substrate for the reaction, respectively. Proteins were confirmed by Coomassie staining in the input panel (bottom). Ubiquitination of KRAS4B was detected by immunoblotting as above (upper). d H460 cells expressing si-Smurf2 and Strep-DX2 were subjected to immunoblotting. Quantitative levels of KRAS were depicted below its blot. e In vitro pull-down assay showing the effect of DX2 on the binding of Smurf2 to KRAS4B. Smurf2, GST-KRAS4B, and DX2 proteins were mixed, and co-precipitation of DX2 and Smurf2 with KRAS4B was monitored. f Inhibitory effect of DX2 on Smurf2-mediated ubiquitination of KRAS4B. 293T cells expressing GFP-KRAS4B, FLAG-Smurf2, and Strep-DX2 were treated with MG-132 and separated into cytosol and membrane fractions. g Analysis of binding kinetics for DX2 or Smurf2 to KRAS (left), and KRAS, HSP70 or p14ARF to DX2 (right) upon EGF signal. All the interactions analyzed by immunoprecipitation (Supplementary Fig. ) were quantified and presented as graphs. C and N indicate cytosol and nucleus, respectively. The maximum blot intensities obtained from immunoprecipitation of each protein pair was taken as 1, and the other blot intensities were divided by the maximum intensity values and presented as the relative values. h Ubiquitination assay validating the Smurf2-dependent ubiquitination sites of KRAS4B. H460 cells expressing GFP-KRAS4B mutants and FLAG-Smurf2 were treated with MG-132 and subjected to the ubiquitination assay. Source data are provided as a . a – f , h , Results are representative of at least three independent experiments.

Article Snippet: Primary antibodies against KRAS and DX2 were purchased from Novus Biologicals and CureBio, respectively.

Techniques: Expressing, Mutagenesis, Western Blot, Ubiquitin Proteomics, In Vitro, Staining, Pull Down Assay, Binding Assay, Membrane, Immunoprecipitation

Journal: Nature Communications

Article Title: AIMP2-DX2 provides therapeutic interface to control KRAS-driven tumorigenesis

doi: 10.1038/s41467-022-30149-2

Figure Lengend Snippet: a Structure of BC-DXI-32982 (DXI hereafter). b Inhibitory effect of DXI on the binding of DX2 and KRAS4B. Binding pair of PRKACA and PRKAR2A was used for checking the binding specificity. N.D. denotes “not determined” in the tested range of dose. The experiment was independently repeated thrice and error bars denote S.D. Data are presented as mean values ± S.D. P value is from the two-sided t test. c Endogenous immunoprecipitation showing the interaction between DX2 and KRAS in H460 cells treated with DXI. Results are representative of at least three independent experiments. d In vitro pull-down assay showing direct inhibition of DXI on the binding of DX2 and KRAS4B. DXI was incubated with the mixture of cellular extracts expressing GFP-KRAS4B and the purified GST-DX2 proteins. Results are representative of at least three independent experiments. e Suppression of KRAS level via DXI. H460 cells treated with various concentration of DXI for 18 h were subjected to immunoblotting and RT-PCR. Results are representative of at least three independent experiments. f Ubiquitination assay analyzing DXI-mediated ubiquitination of KRAS. Results are representative of at least three independent experiments. g Heat map showing EC 50 of DXI on lung, colorectal, and pancreatic cancer cell lines expressing different levels of DX2 (Supplementary Fig. ). In each type of cancers, the maximum and minimum EC 50 values were represented as blue color with the highest and lowest intensity, respectively, and the rests were graded according to their relative values. h , i In vivo efficacy of DXI on tumor growth. Mice ( n = 3) xenografted with H460 cells were intraperitoneally injected with DXI (1 or 5 mg/kg) five times in 12 d ( h ). Mice ( n = 6) xenografted with HCC1588 or A549 cells were intravenously injected with DXI (5 mg/kg) five times during the experiment ( i ). All error bars represent the standard deviation (S.D.). P value is from the two-sided t test. Source data are provided as a .

Article Snippet: Primary antibodies against KRAS and DX2 were purchased from Novus Biologicals and CureBio, respectively.

Techniques: Binding Assay, Immunoprecipitation, In Vitro, Pull Down Assay, Inhibition, Incubation, Expressing, Purification, Concentration Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Ubiquitin Proteomics, In Vivo, Injection, Standard Deviation

Journal: Nature Communications

Article Title: AIMP2-DX2 provides therapeutic interface to control KRAS-driven tumorigenesis

doi: 10.1038/s41467-022-30149-2

Figure Lengend Snippet: a In vitro pull-down assay showing the direct binding of DXI with DX2, but not with KRAS4B. Proteins co-precipitated with Biotin-DXI #2 (Bio-DXI) were determined by SDS-PAGE and Coomassie staining. Biotin was used as a negative control. Results are representative of at least three independent experiments. b In vitro pull-down assay showing the specific interaction of compound with DX2 using Bio-DXI and DXI. Results are representative of at least three independent experiments. c Binding mode of DXI to DX2 as obtained from the MD simulation. The representative structure at 371.2 ns is shown as an electrostatic surface model (first from above). Zoomed-in view of the detailed interactions is presented and binding residues of the compound (green) were depicted as a stick model (second from above). MD snapshots of DX2 and KRAS4B complex at 533.7 ns (third from above). Overlay of above two MD snapshots by superimposing over DX2. The dashed circle denotes the contact region proposed to be responsible for the DXI-mediated inhibition of the interaction of DX2 with KRAS4B (bottom). d Graphs showing the critical residues of DX2 for binding with DXI. Results from the in vitro binding of DX2 mutants with DXI; compound-mediated change in KRAS levels and inhibition of DX2 mutants-KRAS4B were quantified (Supplementary Fig. ). e Anchorage-independent colony formation assay validating the significance of the binding between DXI and DX2. Representative images are shown (bottom). The experiment was independently repeated thrice and error bars denote S.D. Data are presented as mean values ± S.D. P value is from the two-sided t test. Source data are provided as a .

Article Snippet: Primary antibodies against KRAS and DX2 were purchased from Novus Biologicals and CureBio, respectively.

Techniques: In Vitro, Pull Down Assay, Binding Assay, SDS Page, Staining, Negative Control, Inhibition, Colony Assay

Journal: PLOS ONE

Article Title: The KRAS, ATR and CHEK1 expression levels in endometrial cancer are the risk factors predicting recurrence

doi: 10.1371/journal.pone.0302075

Figure Lengend Snippet: The primer sequences used in the study.

Article Snippet: IHC was performed using primary antibodies against KRAS (Thermo Fisher Scientific, USA), phosphorylated (p) ATR (Ser428) (Thermo Fisher Scientific, USA), phosphorylated (p) CHEK1 (Ser296) (Thermo Fisher Scientific, USA).

Techniques:

Journal: PLOS ONE

Article Title: The KRAS, ATR and CHEK1 expression levels in endometrial cancer are the risk factors predicting recurrence

doi: 10.1371/journal.pone.0302075

Figure Lengend Snippet: The mRNA expression levels of KRAS , ATR , CHEK1 genes in endometrial cancer tissue samples at stage I with and without recurrence in anamnesis.

Article Snippet: IHC was performed using primary antibodies against KRAS (Thermo Fisher Scientific, USA), phosphorylated (p) ATR (Ser428) (Thermo Fisher Scientific, USA), phosphorylated (p) CHEK1 (Ser296) (Thermo Fisher Scientific, USA).

Techniques: Expressing

Journal: PLOS ONE

Article Title: The KRAS, ATR and CHEK1 expression levels in endometrial cancer are the risk factors predicting recurrence

doi: 10.1371/journal.pone.0302075

Figure Lengend Snippet: Representative microphotographs of KRAS (a), pATR (b) and pCHEK1(c) protein detection. (A), (B)—low differentiated ECE (× 400); (C)—moderately differentiated ECE (× 400). Immunohistochemical staining, additional staining by Mayer’s hematoxylin.

Article Snippet: IHC was performed using primary antibodies against KRAS (Thermo Fisher Scientific, USA), phosphorylated (p) ATR (Ser428) (Thermo Fisher Scientific, USA), phosphorylated (p) CHEK1 (Ser296) (Thermo Fisher Scientific, USA).

Techniques: Immunohistochemical staining, Staining

Journal: Frontiers in Oncology

Article Title: Identification and Confirmation of the miR-30 Family as a Potential Central Player in Tobacco-Related Head and Neck Squamous Cell Carcinoma

doi: 10.3389/fonc.2021.616372

Figure Lengend Snippet: The tumor growth might be suppressed by miR-30b-5p or miR-30e-5p. (A) Injection protocols of the miR-30b-5p or miR-30e-5p for subcutaneous tumorigenesis in nude mice. (B) Macro images of subcutaneous tumors. (C, E) The volume of subcutaneous tumors in nude mice. (D, F) The body weight of tumorigenesis nude mice. (G) Representative images of H&E staining of subcutaneous tumors injected with different miRNAs (400×). (H, I) The expression of KRAS or Ki67 in subcutaneous injected with different miRNAs (400×). (** p <0.01, *** p <0.001).

Article Snippet: The sections were analyzed using primary antibodies against KRAS (1:800, rabbit, Proteintech, China), Ki67 (1:2000, rabbit, Proteintech, China) and a biotin-conjugated goat anti-rabbit polyclonal antibody (1:50; ZSGB-BIO, China) as the secondary antibody.

Techniques: Injection, Staining, Expressing