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. 2024 Jun 26;13:RP94605. doi: 10.7554/eLife.94605

Figure 5. Fak loss inhibits ERK phosphorylation and upregulates Lgr4.

(A andB) Immunoblotting analysis of intestinal mucosa lysates from indicated bowel subsites in indicated 6-week-old mice. (C) Immunoblotting analysis of cecum lysates from 6-week-old BC mice treated with vehicle or EGFR inhibitor erlotinib for 4 hr. Each lane represented a single mouse. (D) Immunoblotting analysis of cecum lysates from 6-week-old BC mice treated with vehicle or FAK inhibitor PF-562271 for 4 hr. Each lane represented a single mouse. (E) Immunoblotting analysis of lysates from freshly isolated cecal crypts and cecal organoids treated with DMSO, MEK inhibitor PD0325901, or erlotinib, respectively as described in Methods. (F) qRT-PCR of Lgr4 using lysates from HT-29 cells treated with the vehicle and MEKi for 4 hr. Data presented as mean ± SD (***p<0.001; Student’s t-test, two-tailed). (G) qRT-PCR of Lgr4 using cecum lysates from BC mice treated with vehicle or MEKi for 6 hr. Data presented as mean ± SD (**p<0.01; Student’s t-test, two-tailed). Abrogation of ERK phosphorylation at T202/Y204 in the cecum was confirmed by western blot. (H) qRT-PCR of Lgr4 in cecum from BC and FBC mice (n=3 per group). Data presented as mean ± SD (p value calculated using two-tailed Student’s t-test). (I) Immunoblotting analysis of the lysates from HT-29 cells treated with cycloheximide (100 μg/ml) and/or MEK inhibitor PD0325901 (10 μM) as indicated.

Figure 5—source data 1. Uncropped and labelled gels for (Figure 5).
Figure 5—source data 2. Raw unedited gels for (Figure 5).

Figure 5.

Figure 5—figure supplement 1. Fak loss downregulates BRAFV600E-induced ERK phosphorylation.

Figure 5—figure supplement 1.

(A) Immunoblotting analysis of cecal lysates from indicated 6-week-old mice. (B) qRT-PCR of selected ERK transcriptional output markers in cecum from vehicle- and erlotinib-treated BC mice (n=3 per group). Data presented as mean ± SD (ns, not significant; *p<0.05; Student’s t-test, two-tailed). (C) The cecal mucosa lysates from 6-week-old Ptk2fl/fl and Vil1-Cre;Ptk2fl/fl mice were used for immunoblotting. Each lane represented a single mouse. (D) The cecal mucosa lysates from 6-week-old vehicle-treated B and BC mice and EGFR inhibitor erlotinib-treated BC mice were used for immunoprecipitation and immunoblotting with the indicated antibodies. (E) The lysates from HT-29 cells treated with vehicle, EGFR inhibitor erlotinib, or FAK initiator PF-562271 were used for immunoprecipitation and immunoblotting with the indicated antibodies.
Figure 5—figure supplement 1—source data 1. Uncropped and labelled gels for (Figure 5—figure supplement 1).
Figure 5—figure supplement 1—source data 2. Raw unedited gels for (Figure 5—figure supplement 1).
Figure 5—figure supplement 2. qRT-PCR of selected ERK transcriptional output markers in cecum from vehicle- and MEKi-treated BC mice (n=3 per group).

Figure 5—figure supplement 2.

Data presented as mean ± SD (ns, not significant; *p<0.05; **p<0.01; ***p<0.001, Student’s t-test, two-tailed).