Journal: Molecular Oncology

Article Title: SUMOylation modulates the LIN28A‐let‐7 signaling pathway in response to cellular stresses in cancer cells

doi: 10.1002/1878-0261.12694

Figure Lengend Snippet: SUMOylation of LIN28A exacerbates its inhibition of let‐7 biogenesis. (A) Disrupting SUMOylation system interferences let‐7 biogenesis. HeLa‐shpLKO.1, HeLa‐shSENP1, and HeLa‐shUBC9 cells were transiently transfected with HA‐LIN28A. 48 h after transfection, the expression levels of endogenous let‐7a and let‐7c were detected by northern blotting with indicated probes. The knockdown efficiency of SENP1 and UBC9 was detected by western blot with indicated antibodies. The SENP1 and UBC9 bands were quantified by imagej software and normalized with Tubulin. (B) Stable knockout of SENP1 leads to down‐regulation of mature let‐7a levels. 293T cells and 293T SENP1 −/− cells were transiently transfected with HA‐LIN28A. The expression level of endogenous let‐7a was analyzed by northern blotting. The effects of knockout of SENP1 were detected by western blotting with indicated antibodies. (C) SUMO1 modification of LIN28A enhances its inhibition of let‐7s biogenesis. 293T cells were co‐transfected with HA‐LIN28A and human pri‐let‐7c, pre‐let‐7a‐1, or pre‐let‐7g, with or without His‐SUMO1, as indicated. 48 h after transfection, RNAs were extracted and separated on 20% polyacrylamide 8 m urea gels. U6 RNA was used as a control. (D) Truncated forms lacking K15 do not inhibit let‐7a biogenesis. 293T cells were transiently transfected with HA‐LIN28A and truncated forms as indicated. Northern blot was used to measure the expression levels of endogenous let‐7a. (E, F) Mutation K15R of LIN28A blocks its inhibition of let‐7 biogenesis. (E) 293T cells were transiently transfected with HA‐LIN28A or HA‐LIN28A‐K15R, along with pri‐let‐7a‐1 or pri‐let‐7c. (F) Stable DU145, MDA‐MB‐231, and T47D‐shLIN28A expressing the control vector, HA‐LIN28A, or HA‐LIN28A‐K15R were used for northern blotting analyses of let‐7s. The expression levels of LIN28A and LIN28A‐K15R were detected by western blotting with anti‐HA and anti‐LIN28A antibodies. All of let‐7 bands were quantified by imagej software and normalized with U6.

Article Snippet: Mouse‐anti‐GST (66001‐1‐Ig), mouse‐anti‐His (66005‐1‐Ig), and mouse‐anti‐Alpha‐Tubulin (66031‐1‐Ig) were purchased from ProteinTech Group (Rosemont, IL, USA).

Techniques: Inhibition, Transfection, Expressing, Northern Blot, Western Blot, Software, Knock-Out, Modification, Mutagenesis, Plasmid Preparation

Journal: Science Advances

Article Title: The autism susceptibility kinase, TAOK2, phosphorylates eEF2 and modulates translation

doi: 10.1126/sciadv.adf7001

Figure Lengend Snippet: ( A ) Volcano plot of Taok2 knockout/ Taok2 WT phosphoproteome (left, prefrontal cortex; middle, hippocampus; right, ventral striatum). Phosphopeptides containing the TAOK2 kinase-recognition motif pTXX[K/R/H] are in purple and highlight potential direct substrates of TAOK2. Two data points are outside of axis limits. ( B and C ) Immunoblot and quantifications of phosphorylated eEF2 (Thr 56 ) from cortical lysates from Taok2 +/+ and Taok2 −/− mice ( n = 5); * P < 0.05, SEM error bars, unpaired t test. β-Actin was used as a loading control. ( D ) Immunoblot of the TAOK2β IP from nontransfected N2a cells and N2a cells overexpressing WT TAOK2β shows the interaction of TAOK2β with eEF2. ( E ) Immunoblots of in vitro kinase assays using recombinant proteins as depicted in the figure show the direct phosphorylation of eEF2 (Thr 56 ) by TAOK2 kinase. ( F and G ) Immunoblot and quantifications of phospho-eEF2 from N2a cell lysates transfected with WT TAOK2β and its respective control (cells expressing pcDNA3.1-myc-tag). n = 3 per condition from different independent transfection experiments; * P < 0.05, SEM error bars, unpaired t test. ( H and I ) Immunoblot and quantifications of phospho-eEF2 from LCLs lysates with TAOK2 mutation A135P (patient) and the nonaffected father. n = 3 per condition; ** P < 0.05; SEM error bars, unpaired t test. ( J and K ) Immunoblot and quantifications of phospho-eEF2 from N2a cell lysates transfected with TAOK2βA135P and its respective control (cells expressing pcDNA3.1-myc-tag). n = 4 per condition from different independent transfection experiments; SEM error bars, ** P < 0.01 by unpaired t test. β-Actin was used as a loading control.

Article Snippet: One microgram from the recombinant human protein TAOK2 (amino acids 1 to 314) (Signal-Chem, T25-11G-10) or the recombinant human protein eEF2 (MyBioSource, MBS1213669) was subjected to process of denaturation, reduction, alkylation, and digestion and processed for normal proteomics as mentioned previously.

Techniques: Knock-Out, Western Blot, In Vitro, Recombinant, Transfection, Expressing, Mutagenesis