Figure 1. Conditional Kras^LSL alleles with different oncogenic mutations and codon usage.

(A) Schematic of generating and activating Kras^LSL alleles with the first three coding exons fused and encoded by native (nat) versus common (com) codons with either a G12D or Q61R mutation. (B) PCR genotyping of two independently derived mouse embryonic fibroblast (MEF) cultures (two biological replicates) with the indicated Kras alleles in the absence and presence of Cre recombinase (CRE) to detect the unaltered wild-type Kras allele product (WT, 488 bp) and the unrecombined (Kras^LSL*, 389 bp) and recombined (LoxP recombined, 616 bp) Kras^LSL allelic products. Gel images were cropped and color inverted for optimal visualization. Full-length gel images are provided in Figure 1—source data 2. (C) Expression levels, determined by immunoblot with an anti-Kras antibody, RAS activity levels, determined by RBD pull-down (RBD-PD) of lysates collected from MEF cells derived from mice with the indicated Kras alleles in the presence of Cre recombinase (CRE). MEF cultures derived from Kras^{LSL-natG12D/+} mice in the absence of Cre recombinase were used as negative control. MEF cultures were either serum starved overnight (starved) or serum starved overnight followed by serum stimulation for 5 min (stimulated). 20% of the elute from RBD-PD and 30 μg total protein from the total cell lysates were loaded. Tubulin serves as loading control. One of two biological replicates; see Figure 1—figure supplement 3 for the second biological replicate. Full-length gel images are provided in Figure 1—source data 3.

Figure 1—figure supplement 1. Codon usage of the four novel Kras^LSL alleles.

Codon usage index versus codon position of Kras with native (nat, blue line), common (com, red line), and rare (rare, orange line) codons. Maximum rare (all rare, gray dotted line) and common (all common, solid black line) codon usage is shown for reference.

Figure 1—figure supplement 2. Expression and activity level of proteins encoded by cDNA versions of the four Kras^LSL alleles.

Expression levels, determined by immunoblot with an anti-FLAG antibody, and activity levels, determined by RBD pull-down (RBD-PD) followed by (A, B) immunoblots of lysates derived from HEK-HT cells transiently expressing the indicated FLAG-tagged Kras proteins and (C, D) by ELISA (n=2). (A, B) and (C, D) are two biological replicates. Tubulin and empty vector (EV) serve as loading and negative controls, respectively. Full-length gel images are provided in Figure 1—source data 1.

Figure 1—figure supplement 3. Second replicate of RBD pull-down assays from Figure 1C.

RBD pull-down (RBD-PD) of lysates of mouse embryonic fibroblast (MEF) cells derived from the indicated Kras^LSL alleles in the presence of Cre recombinase (CRE) after serum starvation overnight (Starved) or then stimulated with serum for 5 min (Stimulated). RBD-PD was performed with two serial dilutions of 500 μg (top) and 200 μg (bottom) MEF lysates. Lysates of Kras^{LSL-natG12D/+} MEFs in the absence of Cre recombinase serve as negative controls. Full-length gel images are provided at Figure 1—source data 3.

Figure 1—figure supplement 4. Expression and activity levels in the lung upon activating each Kras^LSL allele.

(A) Schematic of the experimental design. Two mice from cohorts of Kras^{LSL-natG12D/+}, Kras^{LSL-natQ61R/+}, Kras^{LSL-comG12D/+}, and Kras^{LSL-comQ61R/+} in a Rosa26^CreERT2/+ background, injected with tamoxifen at six to eight weeks of age, seven days later the lungs were removed at the time of necropsy, and protein lysates were subjected to the RBD pull-down (RBD-PD) assay. Two Rosa26^CreERT2/+ mice were treated similarly as a control. (B) RAF1-RBD pull-down (RBD-PD) of lysates of lungs from the indicated Kras^LSL alleles after tamoxifen injection. 20% of the RBD pull-downs were immunoblotted for Kras. Immunoblots of two separate pull downs from total lung lysates are shown as RBD-PD 1 and RBD-PD 2. 30 μg total protein was loaded in each lane as input. (C) The ratio of median Kras activity levels determined by densitometry in each genotype in comparison to the Kras^natG12D/+ allele, calculated from both technical and biological replicates in (B). p-Values calculated by one-way ANOVA multiple pairwise comparisons with Tukey testing (C). ***p<0.001, ****p<0.0001. Full-length gel images are provided in Figure 1—source data 4.

Figure 1—figure supplement 5. qRT-PCR validates an increase in RAS target gene expression with increased Kras activity.

qRT-PCR validates an increase in RAS target gene expression with increased Kras activity. Two mice from cohorts of Kras^{LSL-natG12D/+}, Kras^{LSL-natQ61R/+}, Kras^{LSL-comG12D/+}, and Kras^{LSL-comQ61R/+} in a Rosa26^CreERT2/+ background, injected with tamoxifen at six to eight weeks of age and RNA was extracted from lungs seven days later. Two Rosa26^CreERT2/+ mice were treated similarly as a control (A). First-strand cDNA synthesis and qRT-PCR of selected genes downstream ERK/MAPK pathway were performed using GoTaq 2-Step RT-qPCR kit (B, C). All measurements were normalized against Actin as the internal control using the 2^-ΔΔCt method (n=3). (B, C) Biological replicates. Measurements are provided in Figure 1—source data 5.

Figure 1—figure supplement 6. RPPA analysis of proteins and phosphoproteins within the MAPK signaling in the lung upon activation of Kras^{LSL-natG12D/+} versus Kras^{LSL-comQ61R/+}.

Heatmap of the log₂ fold change of the average abundance of protein and phosphorylated proteins within the MAPK pathway via RPPA analysis upon activation of the Kras^{LSL-natG12D/+} and Kras^{LSL-comQ61R/+} alleles in comparison to the wild-type Kras allele. Data is representative of three technical replicates from three biological replicates.