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. 2019 Jun 18;116(27):13330–13339. doi: 10.1073/pnas.1902658116

Fig. 5.

Fig. 5.

SHOC2-independent ERK activation requires CRAF. (A) KO of individual RAF isoforms does not affect ERK pathway activation by EGF. Serum-starved DLD-1 parental (P) or ARAF, BRAF, and CRAF KO cells generated by CRISPR were stimulated with 25 ng/mL EGF. (B) ERK pathway activation by EGF in CRAF-only or BRAF-only double KO DLD-1 cells (generated by second round CRISPR of BRAF or CRAF respectively in ARAF KO cells) is normal. However, KD of CRAF in ARAF/BRAF KO (CRAF-only) cells inhibits ERK activation. DLD-1 cells transfected with SCR or CRAF siRNAs were stimulated with EGF as before. (C) CRAF KD (but not ARAF or BRAF) inhibits delayed ERK activation in SHOC2 KO cells. DLD-1 parental and SHOC2 KO cells transfected with ARAF, BRAF, or CRAF siRNAs, stimulated with 25 ng/mL EGF and lysates probed by Li-COR. (D) Li-COR quantification of P-MEK and P-ERK in C (mean ± SD) (n = 2). (E) CRAF KD inhibits growth in SHOC2 KO cells. Parental and two SHOC2 KO clones transfected with SCR or A/B/CRAF siRNAs were used for colony formation assays.