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. 2023 Jul 17;14(7):445. doi: 10.1038/s41419-023-05944-4

Fig. 4. BCKDK competitively interacts with talin1 to disrupt TRIM21 binding to talin1 and inhibit talin1 ubiquitination degradation.

Fig. 4

A The expression of talin1 was examined by western blot in HEK293T cells. B The expression of talin1 was examined by western blot in MDA-MB-231 cells. C Exogenous talin1 coimmunoprecipitated with TRIM21 in HEK293T cells. D The degradation protein level of talin1 detected in M231/Ctrl and M231/TRIM21 cells with CHX treatment (100 μg/mL) for indicated time intervals. E Co-IP shows that HA-ubiquitin is pulled down by talin1 in HEK293T cells. HEK293T cells were transfected with Myc-tagged talin1 and HA-ubiquitin, plus Flag-tagged TRIM21 or the vector controls. At 48 h post-transfection, cells were treated with MG132 for 4 h, and then lysates of these cells were subjected to IP with anti-Myc-conjugated beads. Immunoprecipitates were subjected to IB analysis for ubiquitinated talin1 with anti-ubiquitin or for indicated proteins. F HEK293T cells were transfected with Myc-tagged talin1 and HA-ubiquitin, plus Flag-tagged TRIM21, Flag-tagged BCKDK or their vector controls. At 48 h post-transfection, cells were lysed and subjected to IP with anti-Myc-conjugated beads. The immunoprecipitates were subjected to IB analysis for ubiquitinated talin1 with anti-ubiquitin or for indicated proteins. G IP analysis using anti-Myc antibody followed by IB analysis to detect HA-BCKDK and Flag-TRIM21 expression in HEK293T cells.