chx chase assay  (Cell Signaling Technology Inc)

Journal: Signal Transduction and Targeted Therapy

Article Title: Inhibin beta A drives colorectal cancer progression through macrophage M2 polarization and mitochondria-dependent ferroptosis suppression

doi: 10.1038/s41392-025-02518-y

Figure Lengend Snippet: INHBA acts as a scaffold protein to prevent the TRIM21-driven ubiquitination and degradation of SLC25A10. a Western blot of SLC25A10 in CRC cells with INHBA silenced or overexpressed after MG132 treatment (20 μM, 4 h). b CHX chase (50 μg/ml, TargetMol, USA) of INHBA-knockdown CRC cells; SLC25A10 abundance determined by Western blot at 0, 3, 6, 9 h. c IP was performed using anti-Flag to detect INHBA-interacting proteins. d IP was performed using anti-HA to identify TRIM21-interacting proteins. e IP was were performed using anti-HA to detect SLC25A10-interacting proteins. f IP was performed using anti-HA to identify TRIM21-interacting proteins. g Enrichment and detection of SLC25A10-interacting proteins: CRC cells were transfected with HA-TRIM21 together with Myc-SLC25A10, Flag-INHBA, or an empty vector. 48 h post-transfection, cells were exposed to MG132 (20 μM, 4 h). Myc-SLC25A10 was immunoprecipitated with anti-Myc beads, and immunoprecipitates were detected via Western blot with anti-HA. h Detection of SLC25A10 ubiquitination levels: CRC cells were transfected with Myc-SLC25A10 and HA-Ub together with Flag-INHBA, His-TRIM21, or an empty vector. 48 h post-transfection, cells were exposed to MG132 (20 μM, 4 h). Myc-SLC25A10 was immunoprecipitated with anti-Myc beads, and ubiquitination was detected by anti-Ub Western blot. i Detection of SLC25A10 ubiquitination levels (K48 and K63): CRC cells were transfected with Myc-SLC25A10 together with HA-Ub-K63, HA-Ub-K48, Flag-INHBA, or empty vector. 48 hours post-transfection, cells were exposed to MG132 (20 μM, 4 h). Myc-SLC25A10 was immunoprecipitated with anti-Myc beads, and ubiquitination was detected by anti-Ub Western blot. j Detection of SLC25A10 ubiquitination levels (K48 linkage): HA-Ub-K48 and Myc-SLC25A10 were co-transfected into CRC cells together with Flag-INHBA, His-TRIM21, or empty vector. After 48 h, cells were exposed to MG132 (20 μM, 4 h). Myc-SLC25A10 was immunoprecipitated with anti-Myc beads, and ubiquitination was detected by anti-Ub Western blot

Article Snippet: Fig. 6 INHBA acts as a scaffold protein to prevent the TRIM21-driven ubiquitination and degradation of SLC25A10. a Western blot of SLC25A10 in CRC cells with INHBA silenced or overexpressed after MG132 treatment (20 μM, 4 h). b CHX chase (50 μg/ml, TargetMol, USA) of INHBA-knockdown CRC cells; SLC25A10 abundance determined by Western blot at 0, 3, 6, 9 h. c IP was performed using anti-Flag to detect INHBA-interacting proteins. d IP was performed using anti-HA to identify TRIM21-interacting proteins. e IP was were performed using anti-HA to detect SLC25A10-interacting proteins. f IP was performed using anti-HA to identify TRIM21-interacting proteins. g Enrichment and detection of SLC25A10-interacting proteins: CRC cells were transfected with HA-TRIM21 together with Myc-SLC25A10, Flag-INHBA, or an empty vector.

Techniques: Ubiquitin Proteomics, Western Blot, Knockdown, Transfection, Plasmid Preparation, Immunoprecipitation