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. 2021 Nov 12;10:e68544. doi: 10.7554/eLife.68544

Figure 1. Protein constituents of native transient receptor potential melastatin-subfamily member 7 (TRPM7) channels identified by multi-epitope antibody-based affinity purification (ME-AP) proteomics.

(A) Topology and localisation of the anti-TRPM7 antibodies used for ME-APs. Established hallmark domains of TRPM7 are colour-coded, TRP (transient receptor potential domain, brown), CC (coiled-coil domain, red), kinase (kinase domain, yellow), SD (serine/threonine-rich substrate domain of kinase(s), green). (B) Upper panel: Two-dimensional gel separation of TRPM7 channels in CL-47 solubilised membrane fractions of HEK293 cells with (upper panel) or without (lower panel) transfection of HA-tagged Trpm7, Western-probed with an anti-TRPM7 antibody (Materials and methods). Size (blue native polyacrylamide gel electrophoresis [BN-PAGE]) and molecular weight (SDS-PAGE) are as indicated. Lower panel: Abundance-mass profile of TRPM7 obtained by cryo-slicing blue native mass spectrometry (csBN-MS) in a CL-47 solubilised membrane fraction from adult mouse brain (a total of 192 gel slices). Inset: Abundance of the indicated proteins in the mouse brain. Note the large apparent molecular mass of the native TRPM7 channel in both culture cells and mouse brain, markedly exceeding the mass calculated for tetrameric channel assemblies (about 850 kDa, red circles). (C) Table summarising the results of all anti-TRPM7 APs performed with the indicated antibodies on membrane fractions prepared from rodent brain and cultured HEK293 cells. Solubilisation conditions and specificity of purification of the listed proteins determined by comparison with stringent negative controls are colour-coded as given in the upper left; MW is indicated on the right. TUC refers to series of APs with target-unrelated control antibodies. Note that TRPM7 channels co-assemble with all CNNM family members and ADP-ribosylation factor-like protein 15 (ARL15) in the brain and HEK293 cells.

Figure 1.

Figure 1—figure supplement 1. The specificity of an anti-transient receptor potential melastatin-subfamily member 7 (TRPM7) mouse monoclonal antibody in Western blot assessment of the recombinant TRPM6 and TRPM7 proteins.

Figure 1—figure supplement 1.

0.8 µg/ml of 2C7 IgG (upper panel) or 1.3 µg/ml of 4F9 IgG (lower panel) were used for Western blot analysis of untransfected HEK293 cells (Control 1) or cells transfected with mouse Trpm7 cDNA (mTRPM7), uninduced (Control 2) or induced HEK293-Rex cells expressing the human TRPM7 (hTRPM7), HEK293 cells transfected with human TRPM6 (hTRPM6), or mouse Trpm6 cDNA (mTRPM6). Representative results of two independent experiments are shown.