HA-Tag Antibody - #T0008

Figure 6. YTHDF1 regulates EIF3C translation in an m6A-dependent manner. (A) Relative RNA level of EIF3C in A2780 and SKOV3 upon YTHDF1 knockdown. (B) Western blot detected the protein level of EIF3C in A2780 and SKOV3 cells upon YTHDF1 knockdown. (C) Gene-specific m6A qPCR validation of m6A levels in A2780 and SKOV3 cells. Primers to m6A negative region of EEF1A as the negative control and primers to m6A postive region of EEF1A as the positive control. (D) YTHDF1 RIP followed by RT-qPCR confirmed the interaction between YTHDF1 and EIF3C mRNA. (E) Schematic representation of wild-type (YTHDF1-wt) and mutant (YTHDF1-mut) YTHDF1 constructs. (F) RIP-derived RNA and protein in A2780 cells were measured by RT-qPCR and western blot, respectively. GAPDH was used as the negative control in western blot assays. (G) Western blot confirmed HAtagged EIF3C expression in A2780 or SKOV3 cells co-transfected with empty vector, wild-type or mutant Flag-tagged YTHDF1 and wild-type or mutant HA-tagged EIF3C. (H) Nascent protein synthesis was detected by HPG incorporation upon YTHDF1 knockdown or EIF3C knockdown in A2780 cells. Scale bar, 100  m. Data are shown as means ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant.

FIGURE 1| Interaction of OsHRZ1 with OsPRI2 and OsPRI3.（c) CoIP assay. Total proteins from different combinations with OsHRZ1‐GFP and HA‐OsPRI2/HA‐OsPRI3/HA‐GUS were immunoprecipitated with GFP‐Trap followed by immunoblotting with the indicated antibodies. HRZ1‐GFP/HA‐GUS, negative control. Protein molecular weight (in kDa) is indicated.

FIGURE 1 Interaction of OsHRZ1 with OsPRI2 and OsPRI3. (a) Yeast two‐hybrid assays. Yeast co‐transformed with different BD and AD plasmid combinations were spotted in parallel in 10‐fold dilution series on synthetic dropout medium lacking Leu/Trp/His/Ade. The C‐terminal truncated OsHRZ1 and full‐length OsPRI2/3 were cloned into pGBKT7 and pGADT7, respectively. OsHRZ1‐C/OsPRI1, positive control. OsHRZ1‐ C/Empty, negative control. (b) Pull‐down assay. OsHRZ1 was fused with the GST tag and OsPRI2/3 were fused with the His tag. Recombinant proteins were expressed in E. coli. Proteins were pulled down by glutathione Sepharose 4B and detected using the anti‐His antibody. Protein molecular weight (in kDa) is indicated. (c) CoIP assay. Total proteins from different combinations with OsHRZ1‐GFP and HA‐OsPRI2/HA‐OsPRI3/ HA‐GUS were immunoprecipitated with GFP‐Trap followed by immunoblotting with the indicated antibodies. HRZ1‐GFP/HA‐GUS, negative control. Protein molecular weight (in kDa) is indicated. (d) Degradation of OsPRI2 or OsPRI3 was carried out by detecting the OsPRI2/3‐GFP protein level in co‐infiltration experiments with increasing amounts of OsHRZ1‐GFP. GFP proteins were used as an internal control. Anti‐GFP antibody was used in western blot. Protein molecular weight (in kDa) is indicated. Stars indicate the non‐specific bands. Numbers indicate the ratio of the concentrations of agrobacteria used in co‐infiltration. Empty vector, a binary vector pOCA30 with a 35S promoter; GFP, 35S:GFP in pOCA30; OsPRI2‐GFP, 35S:OsPRI2‐GFP in pOCA30; OsPRI3‐GFP, 35S:OsPRI3‐GFP in pOCA30; OsHRZ1‐GFP, 35S:OsHRZ1‐GFP in pOCA30. (e) Cell‐free degradation. Ten‐day‐old roots grown in Fe‐sufficient solution were harvested and used for protein extraction. Incubation with or without MG132 was performed over the indicated time course

Figure 2. Mtf2 and Sls1 are required for the assembly of Mrh5C.A and B, loss of Mtf2 disrupts Mrh5C. Mitochondrial extracts prepared from WT cells expressing untagged Sls1 (negative control) or Sls1-FLAG (positive control), and Δmtf2 cells expressing Sls1-FLAG were subjected to anti-FLAG co-immunoprecipitation (A). Mitochondrial extracts prepared from WT cells expressing untagged Mrh5 or Mrh5-Myc, and Δmtf2 cells expressing Mrh5-Myc were subjected to anti-Myc co-immunoprecipitation (B). C, loss of Sls1 disrupts Mrh5C. WT cells expressing untagged Mrh5 or Mrh5-Myc and Δmtf2 cells expressing Mrh5-Myc were subjected to anti-Myc co-immunoprecipitation. D, loss of Mrh5 does not affect interactions among other members of Mrh5C. WT cells expressing untagged Sls1 or Sls1-FLAG, and Δmrh5 cells expressing Sls1-FLAG were subjected to anti-FLAG co-immunoprecipitation. E, loss of Ppr4 does not affect interactions among other members of Mrh5C. WT cells expressing untagged Sls1 or Sls1-FLAG, and Δppr4 cells expressing Sls1-FLAG were subjected to anti-FLAG co-immunoprecipitation. F, Mtf2 and Sls1 form a complex independently of Mrh5 and Ppr4. WT cells expressing untagged Sls1 or Sls1-FLAG, and Δmrh5Δppr4 cells expressing Sls1-FLAG were subjected to anti-FLAG co-immunoprecipitation. Mitochondrial extracts (IN) and immunoprecipitates (IP) were analyzed by immunoblotting using specific anti-tag Abs. Genes encoding the Mrh5C subunits were endogenously tagged to facilitate their detection.