Fig 1: FILNC1 is highly expressed in kidney and its expression is downregulated in renal cancers. a Bar graph shows the tumor/normal kidney ratios of FILNC1 expression in 23 paired ccRCC and normal kidney samples from the data set generated by Peña-Llopis et al.29 b Bar graph shows the tumor/normal kidney ratios of FILNC1 expression by real-time PCR from 40 matched ccRCC and normal kidney samples. Values represent mean ± s.d. from three independent measures, two-tailed Student’s t-test. c The box plot shows the expression pattern of FILNC1 for ccRCC and normal kidney samples from the TCGA data set. The boxes show the median ± 1 quartile, with whiskers extending to the most extreme data point within 1.5 interquartile range from the box boundaries (n tumor = 449, n normal = 67, Wilcoxon test). d A Kaplan–Meier plot of renal cancer patients stratified by the expression levels of FILNC1 (n high = 312, n low = 134, log-rank test). e, f Scatter plots show the inverse correlation of FILNC1 with ALDOC (e) or PDK1 (f) expression in human renal tumors, respectively
Fig 2: EMD interacted and inhibited PDHA. (A) Related metabolites and kinases in aerobic oxidation and glycolysis. (B‐C) Protein and mRNA level of aerobic oxidation and glycolysis related kinases were measured by IB (B) and qPCR (C) in EMD overexpressed or knockout H2030 cells. (D) Co‐IP experiments were performed using anti‐EMD or anti‐PDHA antibodies in H2030 cells. IgG‐immunoprecipitated or no‐antibody used samples were analysed in parallel. Co‐immunoprecipitated PDHA and EMD expression was measured. (E) Colocalization of EMD and PDHA was measured by IF in H2030 cells. Scale bar, 20 μm. (F) Proximal protein ligation between EMD and PDHA was measured by PLA in H2030 cells. Scale bar, 50 μm. (G) The location of the EMD IT and LEM domain and PDHA phosphorylation sites on their total proteins. (H) Reciprocal co‐IP experiments were performed using anti‐FLAG or anti‐HA antibodies in PDHAWT‐HA overexpressed H2030 cells with or without EMDWT‐FLAG, EMDDel‐LEM‐FLAG or EMDDel‐IT‐FLAG overexpression. (I) PDHA expression and phosphorylation were measured by normal and Phostag‐IB, respectively, in H2030 cells with or without EMDWT‐FLAG, EMDDel‐LEM‐FLAG or EMDDel‐IT‐FLAG overexpression. PDHA and PDK1 interaction was measured by co‐IP using anti‐PDHA followed by IB experiments. The PDHA level in each co‐IP sample was adjusted to the same protein content. (J) Reciprocal co‐IP experiments were performed using anti‐FLAG or anti‐HA antibodies in EMDWT‐FLAG overexpressed H2030 cells with or without PDHAWT‐HA, PDHADel‐220‐240‐HA or PDHADel‐290‐310‐HA overexpression. (K) Relative acetyl‐CoA, pyruvate and PEP level were measured in H2030 cells with or without EMDWT‐FLAG, EMDDel‐LEM‐FLAG or EMDDel‐IT‐FLAG overexpression. (L) EMD and HA expression were measured in H2030 cells with or without indicated WT or mutant PDHA overexpressed. The data are shown as the mean ± SD from three biological replicates (including IB). Data in C and K were analysed using a one‐way anova test. **, P < 0.01, NS, non‐significant
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