Journal: Cell death & disease

Article Title: UBE2S interacting with TRIM21 mediates the K11-linked ubiquitination of LPP to promote the lymphatic metastasis of bladder cancer.

doi: 10.1038/s41419-023-05938-2

Figure Lengend Snippet: Fig. 3 UBE2S interacts with TRIM21 and LPP in both the nucleus and cytoplasm of BCa. A The endogenous interaction among UBE2S, TRIM21 and LPP in BCa cells was determined by performing co-IP and western blot assays. Anti-UBE2S (14115-1-AP, Proteintech), anti-TRIM21 (12108-1-AP, Proteintech) and anti-LPP (25045-1-AP, Proteintech) were used for Co-IP (each 2 μg). B Representative immunofluorescence images of UBE2S, TRIM21 and LPP colocalization in the nucleus and cytoplasm of BCa cells. Scale bars: white, 25 μm. DAPI was used to label the cell nucleus. C UBE2S, TRIM21 and LPP protein levels in UBE2S-silenced or UBE2S-overexpressing BCa cells. D TRIM21 and LPP protein levels in TRIM21-silenced BCa cells.

Article Snippet: Briefly, a 60 μl conjugation reaction containing 5 μg ubiquitin or ubiquitin-K11R, 5 μg LPP, 1 μg UBE1, 1 μg UBE2S, 1 μg TRIM21, 5 mM ATP (D7378, Beyotime) and 5mM MgCl2 (ST269, Beyotime) was incubated at 37 °C for 1 h. Reactions were terminated by adding 4× loading buffer and subsequently analyzed by western blot with an antibody against LPP (25045-1-AP, Proteintech).

Techniques: Co-Immunoprecipitation Assay, Western Blot

Journal: Cell death & disease

Article Title: UBE2S interacting with TRIM21 mediates the K11-linked ubiquitination of LPP to promote the lymphatic metastasis of bladder cancer.

doi: 10.1038/s41419-023-05938-2

Figure Lengend Snippet: Fig. 4 UBE2S interacts with TRIM21 to degrade LPP by inducing K11-linked ubiquitination. A LPP protein levels in different groups of BCa cells after treatment with MG132 (10 μM, 12 h) or DMSO. The total (B, C) and K11-linked (D, E) ubiquitination levels of LPP in UBE2S-knockdown or TRIM21-knockdown HEK293T cells transfected with corresponding plasmids (48 h) and treated with MG132 (10 μM, 12 h). WT wild type. The total (F) and K11-linked (G) ubiquitination levels of LPP in UBE2S- and TRIM21-overexpressing HEK293T cells transfected with corresponding plasmids (48 h) and treated with MG132 (10 μM, 12 h). H An in vitro ubiquitination assay was performed in the presence of LPP, ubiquitin (Ub), Ub-K11R, UBE1, UBE2S and TRIM21. The reaction was analyzed by western blotting with an anti-LPP antibody (25045-1-AP, Proteintech). I Schematic diagram of TRIM21 and its truncated mutant. The total (J) and K11-linked (K) ubiquitination levels of LPP in full-length TRIM21 and TRIM21-ΔRING HEK293T cells transfected with the corresponding plasmids (48 h) and treated with MG132 (10 μM, 12 h).

Article Snippet: Briefly, a 60 μl conjugation reaction containing 5 μg ubiquitin or ubiquitin-K11R, 5 μg LPP, 1 μg UBE1, 1 μg UBE2S, 1 μg TRIM21, 5 mM ATP (D7378, Beyotime) and 5mM MgCl2 (ST269, Beyotime) was incubated at 37 °C for 1 h. Reactions were terminated by adding 4× loading buffer and subsequently analyzed by western blot with an antibody against LPP (25045-1-AP, Proteintech).

Techniques: Ubiquitin Proteomics, Knockdown, Transfection, In Vitro, Western Blot, Mutagenesis