Figure 2
From: The interactome of a family of potential methyltransferases in HeLa cells
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 MaxQuant40 (version 1.5.1.0) and Andromeda. Data filtering and generation of volcano plots was done essentially as described using a one-way ANOVA test42 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.
