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Generation of KRAS knockout clones. (A) Schematic of the <t>CRISPR/Cas9</t> strategy used to generate KRAS knockout 8988T and KP4 cell lines by targeting exon 2 of KRAS . (B) Immunoblot of RAS - less mouse embryonic fibroblasts (MEFs) reconstituted with BRAF V600E , KRAS WT , HRAS WT , or NRAS WT , confirming specificity of the anti-KRAS antibody (clone 3B10-2F2). (C) Immunoblots showing absence of KRAS protein in 8988T (K275, K328) and KP4 (K22, K63) KRAS knockout clones and baseline PI3K (pAKT) and MAPK (pERK1/2) levels relative to parental cell lines. Loading control is HSP90. Images are representative of n = 3 biological replicates. (D) Bar graphs show quantified pERK/ERK and pAKT/AKT levels relative to the parental cell line (8988T or KP4) (mean ± SEM of n = 3 biological replicates) of immunoblots in ( C ). p -values of repeated measures one-way ANOVA with Tukey’s post hoc test are shown.
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Generation of KRAS knockout clones. (A) Schematic of the <t>CRISPR/Cas9</t> strategy used to generate KRAS knockout 8988T and KP4 cell lines by targeting exon 2 of KRAS . (B) Immunoblot of RAS - less mouse embryonic fibroblasts (MEFs) reconstituted with BRAF V600E , KRAS WT , HRAS WT , or NRAS WT , confirming specificity of the anti-KRAS antibody (clone 3B10-2F2). (C) Immunoblots showing absence of KRAS protein in 8988T (K275, K328) and KP4 (K22, K63) KRAS knockout clones and baseline PI3K (pAKT) and MAPK (pERK1/2) levels relative to parental cell lines. Loading control is HSP90. Images are representative of n = 3 biological replicates. (D) Bar graphs show quantified pERK/ERK and pAKT/AKT levels relative to the parental cell line (8988T or KP4) (mean ± SEM of n = 3 biological replicates) of immunoblots in ( C ). p -values of repeated measures one-way ANOVA with Tukey’s post hoc test are shown.
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Generation of KRAS knockout clones. (A) Schematic of the <t>CRISPR/Cas9</t> strategy used to generate KRAS knockout 8988T and KP4 cell lines by targeting exon 2 of KRAS . (B) Immunoblot of RAS - less mouse embryonic fibroblasts (MEFs) reconstituted with BRAF V600E , KRAS WT , HRAS WT , or NRAS WT , confirming specificity of the anti-KRAS antibody (clone 3B10-2F2). (C) Immunoblots showing absence of KRAS protein in 8988T (K275, K328) and KP4 (K22, K63) KRAS knockout clones and baseline PI3K (pAKT) and MAPK (pERK1/2) levels relative to parental cell lines. Loading control is HSP90. Images are representative of n = 3 biological replicates. (D) Bar graphs show quantified pERK/ERK and pAKT/AKT levels relative to the parental cell line (8988T or KP4) (mean ± SEM of n = 3 biological replicates) of immunoblots in ( C ). p -values of repeated measures one-way ANOVA with Tukey’s post hoc test are shown.
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Generation of KRAS knockout clones. (A) Schematic of the <t>CRISPR/Cas9</t> strategy used to generate KRAS knockout 8988T and KP4 cell lines by targeting exon 2 of KRAS . (B) Immunoblot of RAS - less mouse embryonic fibroblasts (MEFs) reconstituted with BRAF V600E , KRAS WT , HRAS WT , or NRAS WT , confirming specificity of the anti-KRAS antibody (clone 3B10-2F2). (C) Immunoblots showing absence of KRAS protein in 8988T (K275, K328) and KP4 (K22, K63) KRAS knockout clones and baseline PI3K (pAKT) and MAPK (pERK1/2) levels relative to parental cell lines. Loading control is HSP90. Images are representative of n = 3 biological replicates. (D) Bar graphs show quantified pERK/ERK and pAKT/AKT levels relative to the parental cell line (8988T or KP4) (mean ± SEM of n = 3 biological replicates) of immunoblots in ( C ). p -values of repeated measures one-way ANOVA with Tukey’s post hoc test are shown.
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Generation of KRAS knockout clones. (A) Schematic of the CRISPR/Cas9 strategy used to generate KRAS knockout 8988T and KP4 cell lines by targeting exon 2 of KRAS . (B) Immunoblot of RAS - less mouse embryonic fibroblasts (MEFs) reconstituted with BRAF V600E , KRAS WT , HRAS WT , or NRAS WT , confirming specificity of the anti-KRAS antibody (clone 3B10-2F2). (C) Immunoblots showing absence of KRAS protein in 8988T (K275, K328) and KP4 (K22, K63) KRAS knockout clones and baseline PI3K (pAKT) and MAPK (pERK1/2) levels relative to parental cell lines. Loading control is HSP90. Images are representative of n = 3 biological replicates. (D) Bar graphs show quantified pERK/ERK and pAKT/AKT levels relative to the parental cell line (8988T or KP4) (mean ± SEM of n = 3 biological replicates) of immunoblots in ( C ). p -values of repeated measures one-way ANOVA with Tukey’s post hoc test are shown.

Journal: bioRxiv

Article Title: Baseline cellular state dictates the molecular impact of KRAS mutant variants in pancreatic cancer cells

doi: 10.64898/2026.03.10.710185

Figure Lengend Snippet: Generation of KRAS knockout clones. (A) Schematic of the CRISPR/Cas9 strategy used to generate KRAS knockout 8988T and KP4 cell lines by targeting exon 2 of KRAS . (B) Immunoblot of RAS - less mouse embryonic fibroblasts (MEFs) reconstituted with BRAF V600E , KRAS WT , HRAS WT , or NRAS WT , confirming specificity of the anti-KRAS antibody (clone 3B10-2F2). (C) Immunoblots showing absence of KRAS protein in 8988T (K275, K328) and KP4 (K22, K63) KRAS knockout clones and baseline PI3K (pAKT) and MAPK (pERK1/2) levels relative to parental cell lines. Loading control is HSP90. Images are representative of n = 3 biological replicates. (D) Bar graphs show quantified pERK/ERK and pAKT/AKT levels relative to the parental cell line (8988T or KP4) (mean ± SEM of n = 3 biological replicates) of immunoblots in ( C ). p -values of repeated measures one-way ANOVA with Tukey’s post hoc test are shown.

Article Snippet: A single-guide RNA (sgRNA) targeting KRAS exon 2 (sequence: 5’-AATTACTACTTGCTTCCTGT-3’) was cloned into the CRISPR-Cas9 expression vector pSpCas9(BB)-2A-GFP (PX458, Addgene plasmid #48138).

Techniques: Knock-Out, Clone Assay, CRISPR, Western Blot, Control