Fig. 2: RPL40 is a primary cytoplasmic substrate of SMYD5 in human cells. | Cell Research

Fig. 2: RPL40 is a primary cytoplasmic substrate of SMYD5 in human cells.

From: SMYD5 is a ribosomal methyltransferase that catalyzes RPL40 lysine methylation to enhance translation output and promote hepatocellular carcinoma

Fig. 2

a Western blot (WB) analysis of SMYD5 in the whole-cell lysate (WCL), cytoplasmic (Cyto), and nuclear fractions (Nucl) from HeLa cells. Lamin B1 and α-Tubulin were used as nuclear and cytoplasmic markers, respectively. b Immunofluorescence (IF) analyses of HA (green), α-Tubulin (red) and DAPI (blue) in Huh7 cells stably carrying HA-SMYD5 expression construct. c In vitro methylation reactions using recombinant GST-SMYD5 or GST (as a control) and cytoplasmic protein lysates from the control and SMYD5 KO1 HeLa cells as substrates. * denotes the candidate substrate signal. d In vitro methylation reactions with recombinant GST-tagged WT, catalytic-dead SMYD5 (Y351A), or GST as control. Cytoplasmic protein lysates from control and RPL40 KD HeLa cells were used as substrates. Top panel, autoradiogram of the methylation assays; middle and bottom panel, WB analyses using the indicated antibodies. e In vitro pull-down assays using recombinant GST-tagged RPL40 and Flag-tagged recombinant SMYD5. f WB analyses of SMYD5, RPL40 K22me3 and RPL40 in the indicated cell lines. SMYD5 KO1 and KO2 cell lines of Huh7 and SMYD5 KO1 cell line of SNU449 were generated by corresponding gRNAs in Materials and Methods. g WB analyses of SMYD5, RPL40 and RPL40 K22me3 in cells. WT HeLa cells were used as the control. SMYD5 KO1 HeLa cells were rescued by overexpressing empty vector, SMYD5 WT, and SMYD5 mutant (Y351A) constructs.

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