Fig. 3: OTUD3 interacts with MYL12A and Rupatadine to stabilize MYL12A. | Cell Death & Disease

Fig. 3: OTUD3 interacts with MYL12A and Rupatadine to stabilize MYL12A.

From: Rupatadine-inhibited OTUD3 promotes DLBCL progression and immune evasion through deubiquitinating MYL12A and PD-L1

Fig. 3

A Flag-labeled wild-type (WT) OTUD3 or OTUD3-C76A was transfected into Farage cells, and cell lysates were analyzed by western blotting (WB) using anti-MYL12A antibody. B Farage cells were transfected with two independent OTUD3 shRNAs, followed by treatment with or without the proteasome inhibitor MG-132 (20 μM, 8 h), and subsequent analysis of OTUD3 and MYL12A was conducted. C In Farage cells transfected with OTUD3 sh-RNA, they were further transfected with Flag-tagged wild-type (WT) OTUD3 or OTUD3-C76A resistant to sh-RNA (sh-Res), WB analysis was performed for MYL12A levels. D In Farage cells stably expressing OTUD3 shRNA, quantitative analysis of MYL12A levels relative to GAPDH was demonstrated by WB after treatment with cycloheximide (CHX, 10 µg/ml). E Farage cells transfected with the specified plasmid were treated with CHX and collected at designated time points for WB, and the half-life of MYL12A was analyzed. F HEK293T cells were transfected with either His-MYL12A alone or in combination with Flag-tagged WT OTUD3 or OTUD3-C76A. Following immunoprecipitation (IP) with Flag beads from cell lysates, WB analysis was conducted using antibodies targeting His and Flag. (From left to right: Empty vector (Vector), OTUD3-WT, OTUD3-C76A, post-transfection with OTUD3-WT followed by Rupa treatment). G Co-immunoprecipitation (CO-IP) was used to confirm the interaction between OTUD3 and MYL12A in Farage cells. H Immunofluorescence revealed the colocalization of OTUD3 and MYL12A in Farage cells pre-fixed on glass slides. I Schematic representation of full-length (FL) OTUD3, MYL12A, and various deletion mutants. J HEK293T cells were co-transfected with His-MYL12A and Flag-tagged FL OTUD3 or deletion mutants. After immunoprecipitation with Flag beads from cell lysates, WB analysis was conducted using antibodies against His and Flag. K HEK293T cells were co-transfected with Flag-OTUD3 and His-tagged FL MYL12A or deletion mutants. After immunoprecipitation with His beads from cell lysates, WB analysis was performed using antibodies against Flag and His. L The synthetic pathway illustrates the process of substituting boronamine for the chlorine group on rupatadine and incorporating biotin labeling. M In vitro pulldown& WB: Biotin-pulldown and WB analysis performed after incubating biotinylated Rupatadine with Farage cell lysates. In situ pulldown & WB: Biotinylated Rupatadine was incubated with Farage cells, followed by cell lysis, biotin pulldown, and subsequent WB analysis. N WB analysis was conducted to assess the outcomes of the in vitro pulldown. O WB analysis was conducted to assess the outcomes of the in situ pulldown. P HEK293T cells were transfected with Flag-tagged FL OTUD3 or deletion mutants, followed by treatment with labeled rupatadine. After biotin-pulldown from cell lysates, WB analysis was conducted using antibodies against Flag. Error bars represent the mean (n = 3) ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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