Fig. 5
Identification of TPM2 as a downstream target of Smad4 SUMOylation. Knockdown of TPM2 impairs spatial learning and memory formation. a Animals received Flag-vector, Flag-Smad4WT, or Flag-Smad4K113RK159R transfection and their CA1 tissue was subjected to RT-PCR analysis of TPM2 and HPRT gene expression 48 h later. Plasmid transfection and expression were confirmed by immunoprecipitation and immunoblotting with anti-Flag antibody. b Separate animals received the same plasmid transfections as described in (a) and their CA1 tissue was subjected to RT-qPCR analysis of TPM2 mRNA expression. n = 7 for each group, F(2,18) = 337.56, # P < 0.001; q = 8.56, **P < 0.01 comparing the Flag-vector and Flag-Smad4WT groups; q = 35.23, # P < 0.001 comparing the Flag-vector and Flag-Smad4K113RK159R groups. c The same plasmids were transfected to the rat CA1 area, and direct Smad4 binding to the TPM2 promoter was determined by ChIP PCR assay. Plasmid transfection and expression were confirmed by western blotting using the anti-Flag antibody. Experiments were performed in triplicate. d Animals received control siRNA or TPM2 siRNA transfection and were subjected to water maze learning and the probe trial test. n = 9 for each group, F(1,16) = 33.31, # P < 0.001; q = 5.77, # P < 0.001 between the control siRNA and TPM2 siRNA groups for spatial acquisition and t(1,16) = 2.98, **P < 0.01 for the probe trial test. A representative swim pattern from each group is also shown. The statistical difference between the control siRNA and TPM2 siRNA groups for a given trial is indicated by the significance signs: **P < 0.01 and # P < 0.001. e Animals (n = 9 for each group) were sacrificed after the probe trial test and their CA1 tissue was subjected to western blot analysis of TPM2 expression. The quantified results are also shown. t(1,16) = 23.69, # P < 0.001. f Different animals were subjected to water maze training for 1, 3, or 5 days. Another group served as swimming controls. They were sacrificed at the end of training and their CA1 tissue was subjected to RT-qPCR analysis of TPM2 mRNA expression. n = 6 for each group, F(3,20) = 46.2, # P < 0.001; q = 4.43, **P < 0.01 comparing the control and 1-day training groups; q = 12.14, # P < 0.001 comparing the control and 3-day training groups; q = 14.69, # P < 0.001 comparing the control and 5-day training groups. g Animals were subjected to water maze training for 5 days or served as swimming controls. They were sacrificed at the end of training and their CA1 tissue was subjected to western blot analysis of TPM2 expression. The quantified result is also shown. n = 5 each group, t(1,8) = 5.52, # P < 0.001. Data are expressed as means ± SEMs. Raw data and statistics are provided as Additional file 9. ChIP chromatin immunoprecipitation, Cont control, IB immunoblotting, IP immunoprecipitation, RT-PCR reverse-transcription polymerase chain reaction, RT-qPCR reverse-transcription quantitative real-time polymerase chain reaction, SEM standard error of the mean