pcr fragment  (Addgene inc)

Journal: Life Science Alliance

Article Title: Oncogenic mutations of KRAS modulate its turnover by the CUL3/LZTR1 E3 ligase complex

doi: 10.26508/lsa.202302245

Figure Lengend Snippet: (A) Schematic representation of the approach used in this study to investigate the proximal proteome of K-Ras. The first step is the generation of a fusion K-Ras APEX2 protein with a flexible linker between K-Ras and APEX2. In the second step, phenol biotin and H 2 O 2 were added to the media to facilitate the biotinylation of proteins proximal to the bait, including effectors, regulators, and interactors of K-Ras and proteins located near K-Ras. The next step included the IP using streptavidin beads to capture biotinylated proteins. Finally, biotinylated proteins were digested, and mass spectrometry was used to identify proximal proteins. (B) Western blot analysis showing ERK, pERK, and a-FLAG in both cell extract and IP (immunoprecipitation) using GST-Raf1-RBD (Activate Ras Detection Kit) 2 and 4 h upon starvation. As an IP-positive control, the lysate was incubated with GTPγS, and for an IP-negative control, the lysate was incubated with GDP. (C) Experiments were carried out in biological triplicates (n = 3). KRAS has a long half-life; the absence of tetracycline will decrease the proximal labelling of the newly expressed KRAS. For the starvation condition, cells were directly moved for 30 min of incubation with phenol biotin and subsequent incubation with H 2 0 2 for 45 s. On the other hand, for the FCS induction condition, cells were starved for 15 h with a subsequent incubation with phenol biotin for 30 min. In the last 10 min, an additional 20% FCS (including biotin phenol) was added to activate the cells. 45 s of H 2 0 2 was used to initiate the biotinylation of the KRAS proximal proteins. (D) Western blot analysis showing ERK, pERK, and a-FLAG in-cell extract upon APEX2 labelling under different environmental conditions, including starvation and FCS induction. (E) Western blot analysis indicated biotinylated proteins (streptavidin, DyLight 488 conjugated) upon streptavidin immunoprecipitation. APEX2 experiments were carried out in three independent biological experiments.

Article Snippet: Then KRAS ORF was PCR amplified and cloned into the pcDNA5/FRT Vector APEX2 plasmid using NEBuilder HiFi DNA Assembly following protocol.

Techniques: Mass Spectrometry, Western Blot, Immunoprecipitation, Positive Control, Incubation, Negative Control

Journal: Life Science Alliance

Article Title: Oncogenic mutations of KRAS modulate its turnover by the CUL3/LZTR1 E3 ligase complex

doi: 10.26508/lsa.202302245

Figure Lengend Snippet: (A) Western blotting reveals the presence of both endogenous KRAS and exogenous KRAS APEX2 fusion proteins (WT, G12D, G13D, and Q61H) in the presence of 1 μg/ml tetracycline. Relative expression of the APEX2-KRAS fusion protein over endogenous KRAS was determined by quantifying bands and plotting the ratio (right panel). (B) Western blot indicates the biotinylated proteins in the presence or absence of different c reagents, including tetracycline, phenol biotin, and H 2 O 2 . K-Ras APEX2 stable cell line was treated with 1 μg/ml tetracycline for 24 h where indicated. Then, cells were treated with phenol biotin and/or H 2 O 2 (where indicated), lysed, and proteins were separated by SDS–PAGE. Streptavidin DyLight 488 conjugated antibody was used to visualise biotinylated proteins. * Represents biotinylated background proteins. (C) Imaging of live cells using Opera Phenix microscopy processed using the Columbus Image Analysis System. Panels 1183 Hoechst 33342 for staining DNA (blue); the green fluorescence signal from GFP (green); Differential Interference Contrast. (D) Western blot analysis showing α-FLAG, endogenous KRAS, and Na, K-ATPase expression upon digitonin cytoplasmic-membrane fractionation of stable KRAS APEX2 WT, G12D, G13D, and Q61H FRT T-Rex-expressing HEK293 cell lines. Cells were incubated with or without 1 μg/ml tetracycline for 24 h. (E) Western blot analysis showing α-FLAG and endogenous KRAS in sucrose gradient fractions upon 15 h of starvation or 15 h of starvation and a subsequent 10-min treatment with 20% FCS.

Article Snippet: Then KRAS ORF was PCR amplified and cloned into the pcDNA5/FRT Vector APEX2 plasmid using NEBuilder HiFi DNA Assembly following protocol.

Techniques: Western Blot, Expressing, Stable Transfection, SDS Page, Imaging, Microscopy, Staining, Fluorescence, Membrane, Fractionation, Incubation

Journal: Life Science Alliance

Article Title: Oncogenic mutations of KRAS modulate its turnover by the CUL3/LZTR1 E3 ligase complex

doi: 10.26508/lsa.202302245

Figure Lengend Snippet: (A, B) Volcano plotting of fold changes and P -values derived from t test statistic for proximal proteins identified in WT KRAS-APEX2 in either starvation (A) or FCS-treated condition (B), in which the beads were used as a control. Known KRAS effectors (red), repressors (green), and receptors (blue) are coloured and labelled. (C) Histogram of proteins with differential protein abundances, reflecting enrichment per KRAS species. (D, E) Heatmap showing the log 2 fold change (FC MUT/WT) of classical RAS effectors (D) and KRAS repressors (E), reflecting differentially enriched proximal proteins in our study. An asterisk (*) indicates that the protein hit meets the criteria of log 2 (mutant/WT FC) > 0.5 or < −0.5 and −log10 ( P -value) > 0.7.

Article Snippet: Then KRAS ORF was PCR amplified and cloned into the pcDNA5/FRT Vector APEX2 plasmid using NEBuilder HiFi DNA Assembly following protocol.

Techniques: Derivative Assay, Mutagenesis

Journal: Life Science Alliance

Article Title: Oncogenic mutations of KRAS modulate its turnover by the CUL3/LZTR1 E3 ligase complex

doi: 10.26508/lsa.202302245

Figure Lengend Snippet: (A) Mass spectrometry data analysis schematic representation. Data were initially analysed in MaxQuant to identify proteins present in our samples. Data were then processed with an interaction scoring algorithm SAINTexpress to provide a score on the probability of a true “interaction” (true proximity protein) with our bait. Finally, true proximity proteins were decided to include any protein with a SAINT score above 0.8 and fold difference higher or equal to four as compared with the beads control. On the other hand, MaxQuant results were further analysed on Perseus, and volcano plots were generated. Different databases, including BioGRID, were integrated to explore the identification of known interactors. Finally, PPI networks were generated through Metascape and Cytoscape. (B) All Ras proximal proteins are identified in each APEX2-KRAS “proximitome.” SAINT probability ≥ 0.8; fold change ≥ 4 over beads control (1,373 proteins). Several KRAS interactome families (receptors, repressors, activators, interactors, classical Ras effectors, “proximitome,” MEK-RAF pathway, PIK3 pathway, TIAM-RAC pathway, RALGDS pathway, RASSF pathway) were explored. (C) Heatmap showing the relative abundance of known KRAS interactors in WT KRAS, G12D, G13D, and Q61H KRAS mutant-APEX2 samples under either starvation or FCS-stimulated conditions. (D) Landscape (blue – previously known; grey – this study) of the KRAS-APEX2 “proximitome” (SAINT score ≥ 0.8) relative to the cellular proteome. Previously known Ras effectors, including receptors, repressors, and activators, are coloured red.

Article Snippet: Then KRAS ORF was PCR amplified and cloned into the pcDNA5/FRT Vector APEX2 plasmid using NEBuilder HiFi DNA Assembly following protocol.

Techniques: Mass Spectrometry, Generated, Mutagenesis