Fig. 7

cFAM124A upregulates CTSL enzyme expression through bridging effect and promotes an increase in the tRXRα protein level. A, Western blot analysis of RXRα expression after 24 h of treatment with PD150606 (200 nM, m-calpain inhibitor) or ZFY-CHO (10 µM, CTSL inhibitor). Representative images are shown (left). B, Indicated PDAC cell lines were incubated with ZFY-CHO and then with CHX for indicated time periods before western blot analysis of RXRα and GAPDH expression. Representative images are shown (left). C, CTSL enzyme‑linked immunosorbent assay (ELISA) in PDAC cells with cFAM124A overexpression or knockout. D, Potential cFAM120A-binding proteins were pulled down in cell lysate by RAP assay; these were incubated with the cFAM124A probe and subsequently visualized by MS and silver staining. E, Venn diagram showing the overlap of potential binding proteins of cFAM124A. F, IGF2BP2 was pulled down by the LacZ probe (control) or cFAM124A probe and cFAM124A mutant. G, IGF2BP2 was pulled down by the CTSL 3ʹ-UTR probe after overexpression of CTSL 3ʹ-UTR. H, Association of cFAM124A with CTSL mRNA on RAP assay. I, IGF2BP2 was pulled down by the CTSL 3ʹ-UTR probe on western blotting in the indicated groups. J, Upper: Schematic of the RNA-binding domain within IGF2BP2 and list of different IGF2BP2 truncation mutants. Lower: Immunoblotting with anti-Flag antibody after RNA pulldown assay in PATU8988T cells using RNA cFAM124A or CTSL mRNA probes. K, Pattern diagram of binding among cFAM124A, CTSL mRNA, and IGF2BP2. L, CTSL mRNA expression in the indicated groups. M, CTSL, Akt, RXRα, p-Akt (Thr308), and p-Akt (Ser473) protein expression in the indicated groups. N, CTSL protein concentrations detected by ELISA in the indicated groups