kras gene fragment  (New England Biolabs)

Journal: bioRxiv

Article Title: Phosphorylation Protects Oncogenic RAS from LZTR1-Mediated Degradation

doi: 10.64898/2026.01.07.698128

Figure Lengend Snippet: A ) Schematic of KRAS oncogenic hotspot mutations and putative serine/threonine phosphorylation sites. B ) Representative FACS data for mNG-KRAS G12D constructs harboring indicated S/T to A mutations XG7, RPMI 8226, and MM.1S MM cells, n=3. C ) Average normalized mNG-KRAS G12D phospho-mutant FACS data. Error bars depict standard deviation, n=3. D ) Western blot analysis for mNG-KRAS, PP1C, and GAPDH in XG7 cells expressing indicated mNG-KRAS mutants and either empty vector (-) or DN PP1C (+), n=3. E ) Western blot analysis of in vitro phosphatase assays performed on mNG-KRAS G12D immunoprecipitated from XG7 cells, n=4. F ) Model depicting PP1C directly dephosphorylating KRAS T148. G ) Enrichment of KRAS G12D+T148A (x-axis) vs. KRAS G12D+T148D (y-axis) BioID2 constructs. H ) Representative images from proximity ligation assay (PLA) (red) of RAS and indicated downstream effectors in XG7 cells. DAPI (blue), wheat germ agglutinin (WGA; green). Scale bar is 10 μm. I ) PLA score of indicated PLA pairs normalized to empty vector condition, **** P<0.0001, n=3.

Article Snippet: Synthetic KRAS gene fragments (Twist Bioscience) were cloned into the mNG–8xlinker–pBMN–LYT2 vector via Gibson assembly (New England Biolabs) (Table S4).

Techniques: Phospho-proteomics, Construct, Mutagenesis, Standard Deviation, Western Blot, Expressing, Plasmid Preparation, In Vitro, Immunoprecipitation, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Phosphorylation Protects Oncogenic RAS from LZTR1-Mediated Degradation

doi: 10.64898/2026.01.07.698128

Figure Lengend Snippet: A ) IP-western blot analysis of ubiquitin binding to indicated ectopically expressed KRAS constructs. B ) Scatter plot of ubiquitin-related genes from the KRAS stability screens in . C ) Venn diagram of ubiquitin-related genes with increased KRAS expression (P≤0.05), KRAS interaction partners from BioID2 studies (≥1.0 log2 fold enrichment, ref: ), and tumor suppressor genes identified in RAS-dependent MM lines (CSS≥0.5, ). D ) LZTR1 CSS for KRAS-dependent (pink) vs. RAS-independent (gray) MM lines. E ) Western blot analysis of RAS, LZTR1, and GAPDH following expression of shCTRL or 2 shRNAs targeting LZTR1 in XG7, RPMI 8226, and MM.1S MM lines. F ) The essential interactome of LZTR1. Average CSS for RPMI 8226 and XG7 (x-axis) plotted against average enrichment for proteins from LZTR1-BioID2 experiments in the same lines (y-axis). Pink shading denotes proteins with ≥1.0 log2 fold enrichment and CSS ≤1; purple are callouts discussed in text. G ) Co-IP of indicated KRAS G12D phospho-mutants with LZTR1 in XG7, RPMI 8226, and MM.1S MM cells. H ) Representative images from PLA (red) of RAS and LZTR1 in XG7 cells expressing indicated KRAS mutants. DAPI (blue), wheat germ agglutinin (WGA; green). Scale bar is 10 μm. I ) PLA score of LZTR1-RAS normalized to empty vector condition, **** P<0.0001, * P<0.01, n=3. J ) Western blot analysis of RAS, pT148-RAS, PPP1R2, LZTR1, and GAPDH following transduction with control, PPP1R2, and/or LZTR1 shRNAs. K ) Bar graph of relative changes in mNG-KRAS expression measured by FACS for indicated mutants in shLZTR1 vs. shCTRL treated cells. L ) Immunoblot of ubiquitin binding following mNG-KRAS pulldown in cells transduced harboring G12D, T148A, T148D, K147R or K147R+T148A mutations with ubiquitin in XG7 cells, n=2.

Article Snippet: Synthetic KRAS gene fragments (Twist Bioscience) were cloned into the mNG–8xlinker–pBMN–LYT2 vector via Gibson assembly (New England Biolabs) (Table S4).

Techniques: Western Blot, Ubiquitin Proteomics, Binding Assay, Construct, Expressing, Co-Immunoprecipitation Assay, Plasmid Preparation, Transduction, Control

Journal: bioRxiv

Article Title: Phosphorylation Protects Oncogenic RAS from LZTR1-Mediated Degradation

doi: 10.64898/2026.01.07.698128

Figure Lengend Snippet: A ) Bar graph of KRAS A146 mutations as a percentage of all KRAS mutations across various tumor types. Hematologic malignancies are highlighted in pink. Adapted from ref: 1 . B,C ) Western blot analysis of RAS, PPP1R2, and GAPDH following transduction with control or PPP1R2 shRNAs ( B ) or indicated ectopically expressed RAS mutants ( C ), n=3. D ) Co-IP of indicated KRAS mutants with PPP1R2 or PP1C in XG7 and AMO1 MM cells, n=3. E ) Western blot analysis of pT148 RAS phosphorylation of endogenous RAS in XG7, RPMI 8226, AMO1, and H1112 MM lines, n=3. F ) Western blot analysis of ubiquitin binding following mNG-KRAS pulldown in H1112 cells harboring G12D, A146T, or A146V, n=3. G ) Quantification of immunoblots of RAS expression normalized to GAPDH from indicated MM lines following a time course of treatment with 10 nM cycloheximide (CHX) for the indicated timepoints (n=2; error bars depict standard deviation; representative blots in Fig. S4G). H ) Western blot analysis of RAS, LZTR1, and GAPDH following transduction with control or LZTR1 shRNAs in XG7, AMO1, and H1112 cells, n=3. I ) Scatter plot showing the percentage of KRAS A146 mutations among all KRAS mutations from (x-axis) versus the LZTR1 CHRONOS score from DepMap. J ) Quantification of immunoblots of RAS expression normalized to GAPDH from indicated cell lines (COAD, blue; PAAD, black; GCB DLBCL, orange; AML, red) following a time course of treatment with 10 nM cycloheximide (CHX) for the indicated timepoints (n=2; error bars depict standard deviation; representative blots in Fig. S4G). K ) Dot plot showing the RAS half-life in indicated cell lines (MM, pink; MM KRAS A146, pink hollow circle; COAD, blue; PAAD, black; GCB DLBCL, orange; AML, red). L,M ) Immunoblot analysis of RAS expression following transduction with control shRNA or LZTR1 shRNAs in adenocarcinoma lines ( L ) and GCB DLBCL lines ( M ), n=3.

Article Snippet: Synthetic KRAS gene fragments (Twist Bioscience) were cloned into the mNG–8xlinker–pBMN–LYT2 vector via Gibson assembly (New England Biolabs) (Table S4).

Techniques: Western Blot, Transduction, Control, Co-Immunoprecipitation Assay, Phospho-proteomics, Ubiquitin Proteomics, Binding Assay, Expressing, Standard Deviation, shRNA

Journal: bioRxiv

Article Title: Phosphorylation Protects Oncogenic RAS from LZTR1-Mediated Degradation

doi: 10.64898/2026.01.07.698128

Figure Lengend Snippet: A ) Schematic for a putative kinase phosphorylating RAS T148. B ) Scatter plot of kinase-related genes from the KRAS stability screens in . C ) Venn diagram of kinase genes with decreased KRAS expression (≥0.5 KRAS stability score), RAS interaction partners from BioID2 studies (≥0.8 log2 fold enrichment, ), and essential genes identified in RAS-dependent MM lines (CSS<-1.0, 19 ). D ) Western blot analysis of RAS expression following treatment with 100 nM of the indicated drugs for 24 and 48 hours in XG7, RPMI 8226, and SKMM1 MM cells, n=5. E ) Evaluation of pT148 RAS phosphorylation in cells treated with MG-132 for 8 hours before lysis and the indicated drugs for 48 hours, n=3. F ) Co-IP of PAK1 and PAK2 with ectopically expressed mNG-KRAS G12D in the indicated MM lines, n=2. G ). Indicated cells expressing listed shRNAs or DN PP1C were treated with a dose titration of RMC-6236. Data shown is the ratio of experiment (listed shRNA or DN PP1C) versus shCTRL. Values shown are the average of 2 experiments. H ) in vitro kinase assay of recombinant PAK1 on recombinant KRAS as a substrate. Western blot analysis of phospho-T148 RAS was used as a readout. Inhibition with FRAX597 was used to demonstrate specificity, n=4. I ) Cell viability in XG7 cells ectopically expressing KRAS G12D or KRAS G12D+T148D and treated with indicated drugs: either 100 nM FRAX527 and/or 8 nM RMC-6236. J ) Tumor volume for MM.1S xenografts treated with vehicle (gray), 3 mg/kg FRAX597 (purple), 5 mg/kg RMC-6236 (pink), or the combination of both drugs (red). K ) Inhibition with FRAX597 primes RAS for degradation.

Article Snippet: Synthetic KRAS gene fragments (Twist Bioscience) were cloned into the mNG–8xlinker–pBMN–LYT2 vector via Gibson assembly (New England Biolabs) (Table S4).

Techniques: Expressing, Western Blot, Phospho-proteomics, Lysis, Co-Immunoprecipitation Assay, Titration, shRNA, In Vitro, Kinase Assay, Recombinant, Inhibition

Journal: bioRxiv

Article Title: Phosphorylation Protects Oncogenic RAS from LZTR1-Mediated Degradation

doi: 10.64898/2026.01.07.698128

Figure Lengend Snippet: A ) CRISPR screening strategy to identify regulators of KRAS protein stability. B ) Volcano plot of average KRAS stability scores (n=3). Significant hits for genes that either decrease (pink) or increase (purple) KRAS expression are indicated. Callouts represent genes identified as RAS interaction partners by mass spectrometry . C ) Venn diagram of overlapping genes from KRAS stability screen (≤-0.8 KRAS stability score and P≤0.05), RAS BioID2 proteomics (≥1.0 log2 enrichment vs. control, from ref: ), and genes essential in RAS-dependent MM cell lines (≤-1.0 CSS, from ref: ). D ) Western blot analysis of RAS, PPP1R2, and GAPDH 3 days after transduction with control shRNA (shCTRL) or PPP1R2-targeting shRNAs in XG7, RPMI 8226, and MM.1S MM lines, n=3. E ) PPP1R2-BioID2 enrichment over empty vector in RPMI 8226 cells. F ) Western blot analysis of RAS, PPP1R2, PP1C, and GAPDH 3 days after transduction with shCTRL, shPPP1R2.1, and/or ectopic expression of DN PP1C, n=3. G ) Comparison of protein expression levels between KRAS (x-axis) and average PP1C (PPP1CA, PPP1CB, PPP1CC, and PPP1CC;PPP1CB) in 115 MM patient tumors. Display line is simple linear regression; R 2 =0.1593, P<0.0001. I ) Model of PPP1R2 and PP1C regulation of KRAS protein expression. Under a normal state, PPP1R2 inhibits PP1C activity. Following PPP1R2 disruption, PP1C activity reduces KRAS levels. In contrast, PP1C disruption increases KRAS expression.

Article Snippet: Briefly, 150 ng of KRAS gene fragment was combined with 1 μL of SnaBI-linearized BioID-2 vector and 4 μL of 2× NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) and incubated for 1 h at 50 °C.

Techniques: CRISPR, Expressing, Mass Spectrometry, Control, Western Blot, Transduction, shRNA, Plasmid Preparation, Comparison, Activity Assay, Disruption