Fig 1: Western blotting and RT-qPCR analyses of the differentially expressed proteins in MCF-7 and MX-1 cells. Knockdown efficiency of KD (siRNA-5) was confirmed via western blotting in (A) MCF-7 and (H) MX-1 cells. Western blotting showed that the relative protein expression levels of (B and I) AGA, (C and J) MT1E and (D and K) TDO2 were increased when ILP-2 expression was knocked down. Tubulin and GAPDH were used as reference proteins. RT-qPCR analysis indicated that the relative mRNA expression levels of (E and L) AGA, (F and M) MT1E and (G and N) TDO2 were increased when ILP-2 expression was knocked down. GAPDH was used as the reference gene. Data are presented as the mean ± SEM and groups were compared with an unpaired Student's t-test (n=3). **P<0.01, ***P<0.001 vs. the NC group. RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA; ILP-2, inhibitor of apoptosis protein-like protein-2; MT1E, metallothionein 1E; AGA, N(4)-(β-N-acetylglucosaminyl)-L-asparaginase; TDO2, tryptophan 2,3-dioxygenase; NC, negative control.
Fig 2: Heatmap and clustering analysis of the expression patterns of DEPs in the KD and NC groups. (A) The heatmap of the DEPs. The table shows the differentially expressed genes in the KD vs. NC group. (B) Sub-clusters 2, 3, 6, 7, 8, 9 and 10 (including 1, 17, 2, 2, 4, 1 and 1 proteins) were upregulated from the NC group to the KD group; sub-clusters 1, 4 and 5 (including 10, 7 and 3 proteins) were downregulated from the NC group to the KD group. DEP, differentially expressed protein; NC, negative control; KD, knockdown; MT1E, metallothionein 1E; AGA, N(4)-(β-N-acetylglucosaminyl)-L-asparaginase; TDO2, tryptophan 2,3-dioxygenase.
Fig 3: Western blot analysis showed that the relative protein expression levels of AGA, MT1E and TDO2 were decreased when the protein expression of ILP-2 was overexpressed. Tubulin was used as the reference protein. Western blot analysis of (A and E) ILP-2, (B and F) AGA, (C and G) MT1E and (D and H) TDO2 in (A-D) MX-1 and (E-H) MCF-7 cells were decreased when ILP-2 was overexpressed. Tubulin was used as a reference protein. Data are presented as the mean ± SEM and groups were compared with an unpaired Student's t-test (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. the NC group. ILP-2, inhibitor of apoptosis protein-like protein-2; MT1E, metallothionein 1E; AGA, N(4)-(β-N-acetylglucosaminyl)-L-asparaginase; TDO2, tryptophan 2,3-dioxygenase; NC, negative control.
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