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 1| Identifcation of the interaction between the NDV M protein and chBRD2 protein.B Identifcation of the interaction between the M protein and chBRD2 protein by co-IP assay. The indicated plasmids were co-transfected into DF-1 cells, and reciprocal co-IP assays were performed to identify the interaction between HA-M and Myc-chBRD2 in DF-1 cells at 36 hpt.

Figure 3. Identification and verification of SENP3-interacting proteins with SENP3–BirA (A) Silver staining of precipitations by BioID. LO2 cells were stably transfected with pCDH–SENP3–BirA (named LO2–SENP3–BirA). LO2–SENP3–BirA cells were untreated or treated with DEN. (B) A pie chart showing the number of proteins identified by MS. (C) The interaction of SENP3 with TKT, NPM1, and ATP5F1A was verified by co-IP. LO2 cells were transfected with pCDH–SENP3–BirA and treated with DEN. (D) The in vitro interaction of SENP3 with TKT was validated by GST pull-down assay. Flag-SENP3 was transfected into HEK293T cells, and SENP3 was purified from cell lysates with anti-Flag M2 gels after 24 h of transfection. GST–TKT was obtained from prokaryotic expression system. (E) The physical interaction of SENP3 with TKT or ATP5F1A was measured by FRET with acceptor photobleaching method. After treatment with DEN for 2 h, LO2 cells were fixed and stained with primary antibodies and fluorescence-conjugated secondary antibodies. Scale bar: 10 μm. The efficiency of FRET from 10 cells are shown. (F) Transient SUMOylated proteins that interact with SENP3 were evaluated. co-IP was performed in HEK293T cells transfected with HA–SENP3.