Journal: Scientific Reports

Article Title: The interactome of a family of potential methyltransferases in HeLa cells

doi: 10.1038/s41598-019-43010-2

Figure Lengend Snippet: Experimental workflow. ( a ) ORFs of METTL proteins were cloned as GFP fusions under the control of a DOX-inducible promoter for targeted single copy integration into HeLa FRT cells. Whole cell extracts and/or nuclear extracts were prepared from GFP-METTL or GFP (control) expressing cells. GFP pull-downs were performed in triplicate followed by mass spectrometry analysis. Analysis of raw data was performed in MaxQuant (version 1.5.1.0) and Andromeda. Data filtering and generation of volcano plots was done essentially as described using a one-way ANOVA test with log2 fold change (FC) > 8 and false discovery rate (FDR) < 0.05 as thresholds. ( b ) Volcano plot of GFP-MBD3 interacting proteins as an example of successful complex identification using this protocol. Significant interactors are indicated. Volcano plots of ( c ) METTL3 and ( d ) METTL14 interactors. The log2 FC of GFP fusions to control in label-free quantification are plotted against the −log10 of the FDR calculated by a permutation-based FDR adapted t-test. The baits are indicated in red.

Article Snippet: GFP, GFP-METTL8 and GFP-METTL16 were purified from stable HeLa FRT cell lines using GFP nanobody sepharose beads (Chromotek).

Techniques: Clone Assay, Control, Expressing, Mass Spectrometry, Quantitative Proteomics

Journal: Scientific Reports

Article Title: The interactome of a family of potential methyltransferases in HeLa cells

doi: 10.1038/s41598-019-43010-2

Figure Lengend Snippet: Confirmation of METTL9 interactor and enzymatic activity of GFP-METTLs. ( a , b ) Validation of interaction between METTL9 and CANX by co-IP. ( a ) CANX is detected in GFP-METTL9 IP (a, lane 5) but not GFP IP (a, lane 2). 10 μl of GFP trap and 2 mg of whole cell extract were used. 20 μg of Input material were loaded for a comparison. ( b ) GFP-METTL9 (left panel, line 2) but not GFP (right panel, line 3) can be detected by immuno-blot with GFP antibody in the CANX IP. 10 μg of calnexin antibody and 4 mg of whole cell extract were used. 200 μg of Input material were loaded for comparison. No antibody (beads alone) used as control. ( c ) In vitro methyltransferase assays demonstrating that our GFP-METTL8 and GFP-METTL16 purifications have the expected RNA methyltransferase activity. GFP (as a control) and GFP-fusion proteins were purified from corresponding DOX-induced HeLa FRT cell lines and used in an in vitro methyltransferase assay on total RNA from HeLa cells as a substrate and 3H-SAM as a methyl-donor. After purification of the RNA, counts per minute (CPM) were quantified by liquid scintillation counting. Ratio of CPM measured for reactions with GFP-METTL fusion proteins relative to GFP control are plotted. Data are shown as mean ± SD from three replicates.

Article Snippet: GFP, GFP-METTL8 and GFP-METTL16 were purified from stable HeLa FRT cell lines using GFP nanobody sepharose beads (Chromotek).

Techniques: Activity Assay, Biomarker Discovery, Co-Immunoprecipitation Assay, Comparison, Control, In Vitro, Purification

Journal: eLife

Article Title: A specific role for importin-5 and NASP in the import and nuclear hand-off of monomeric H3

doi: 10.7554/eLife.81755

Figure Lengend Snippet:

Article Snippet: HeLa FRT cell line (T-REx-HeLa) was sourced from Invitrogen (R71407).

Techniques: Plasmid Preparation, Transfection, Construct, Transformation Assay, Expressing, Sequencing, Blocking Assay, Recombinant, Concentration Assay, Software, Staining