Fig. 5: TRIM29 stabilizes hnRNPA1 by preventing ZFP91-mediated K48 ubiquitination.

A Cells were transfected with a plasmid encoding His-hnRNPA1 and various forms of HA-tagged ubiquitin (including wild-type HA-Ub, K6-, K11-, K27-, K29-, K33-, K48-, or K63-linked Ub) along with either an empty vector or a vector expressing TRIM29 were treated with MG132 (10 μM) for 3 h. The lysates were then subjected to immunoprecipitation using an anti-His antibody and subsequent immunoblotting with an anti-HA antibody. B Cells transfected with either wild-type Ub or the K48R mutant ubiquitin. Then, cell lysates were examined by immunoblotting (IB) using antibodies specific to TRIM29 and hnRNPA1. C Cells were transfected with either the control vector plasmid or a plasmid expressing TRIM29, along with HA-Ub and either wild-type His-hnRNPA1 or its site-specific K-to-R mutant variants. Following transfection, cell lysates were prepared and subjected to IP using anti-His. The precipitated proteins were then analyzed by immunoblotting. D GC cells were transfected with wild-type ubiquitin (HA-Ub) and either wild-type His-hnRNPA1 or the His-hnRNPA1-K8R mutant, and cultured for 72 h in medium containing control shRNA or shRNA targeting TRIM29. After that, cell lysates were harvested and subjected to western blotting for the analysis of hnRNPA1 and TRIM29 protein levels. E Cells were transfected with either the control vector plasmid or a plasmid expressing TRIM29 or ZFP91, along with HA-Ub. Following transfection, cell lysates were prepared and subjected to IP using anti-hnRNPA1. The precipitated proteins were then analyzed by immunoblotting. F Cells were transfected with shRNA targeting ZFP91 or a plasmid expressing ZFP91. Following that, cell lysates were harvested and then analyzed by immunoblotting. G Cells were transfected with shRNA targeting ZFP91 or a plasmid expressing ZFP91 and its mutant ZFP91(C344A/C349A). Following that, cell lysates were harvested and subjected to IP using anti-hnRNPA1. The precipitated proteins were then analyzed by immunoblotting. H Cells were transfected with a plasmid expressing TRIM29 in dose relied manner. Following that, cell lysates were harvested and subjected to IP using anti-hnRNPA1. The precipitated proteins were then analyzed by immunoblotting. I Cells were transfected with shRNA targeting TRIM29 or a plasmid expressing TRIM29. Following that, cell lysates were harvested and subjected to IP using anti-hnRNPA1. The precipitated proteins were then analyzed by immunoblotting. J Cells co-expressing Myc-ZFP91 and His-hnRNPA1 or its deletion mutants were subjected to cell lysis. The precipitated proteins were analyzed by western blotting. *P < 0.05, **P < 0.01, ***P < 0.001.