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. 2023 Mar 22;13:4663. doi: 10.1038/s41598-023-31790-7

Figure 4.

Figure 4

NTSR1-high form is N-terminally cleaved by matrix metalloproteinase to form the NTSR1-low protein form. (A, C, E, G) Arrowheads: Red: NTSR1-high, pink: NTSR1-low, (A) representative anti-NTSR1 WB performed after SDS-PAGE of SDS lysates from HEK293T cells overexpressing NTSR1 (HEK293T-NTSR1) or not (HEK293T-WT). HEK293T-NTSR1 were treated or not (untreated) 1 h with 100 nM Neurotensin alone or cotreated with the broad MMP inhibitor Marimastat. (B) Anti-NTSR1 WB quantification of NTSR1-low and NTSR1-high protein forms of HEK293T-NTSR1 cells presented in A. The NTSR1 signal is normalized to Na + /K + ATPase signal, before normalization to NTS alone treatment condition. + /- SEM, N = 3. (C) representative anti-NTSR1 WB performed after SDS-PAGE of SDS lysates from HEK293T cells overexpressing NTSR1 (HEK293T-NTSR1) or not (HEK293T-WT). HEK293T-NTSR1 were treated or not (untreated) 1 h with 100 nM Neurotensin alone or cotreated with the broad MMP inhibitor Batimastat. (D) Anti-NTSR1 WB quantification of NTSR1-low and NTSR1-high protein forms of HEK293T-NTSR1 cells presented in C. The NTSR1 signal is normalized to Na + /K + ATPase signal, before normalization to NTS alone treatment condition. + /- SEM, N = 3. *p < 0.05; **p < 0.01; ***p < 0.001. (E) representative anti-NTSR1 WB performed after SDS-PAGE of total GPCR fraction extracted from total membranes of HT29-KD (NTSR1 depleted) or HT29#3 (NTSR1-expressing) cells. HT29#3 were treated with vehicle or the broad MMP inhibitor Batimastat alone. Non-saturating higher exposure of the NTSR1-high signal is displayed. (F) Anti-NTSR1 WB quantification of NTSR1-low and NTSR1-high protein forms of HT29#3 cells presented in E. The NTSR1 signal is normalized to Na + /K + ATPase signal, before normalization to vehicle condition. + /- SEM, N = 3. (G) representative anti-NTSR1 WB performed after SDS-PAGE of total GPCR fraction extracted from total membranes of HT29#3 (NTSR1-expressing) cells. HT29#3 were treated with vehicle or the broad MMP inhibitor Marimastat alone. Non saturating higher exposure of the NTSR1-high signal is displayed. (H) Anti-NTSR1 WB quantification of NTSR1-low and NTSR1-high protein forms of HT29#3 cells presented in G. The NTSR1 signal is normalized to Na + /K + ATPase signal, before normalization to vehicle condition. + /- SEM, N = 3. *p < 0.05; **p < 0.01; ***p < 0.001. Uncropped WB blots images are included in Supplementary information.