Journal: The European Journal of Neuroscience

Article Title: MAP6 interacts with Tctex1 and Ca v 2.2/N‐type calcium channels to regulate calcium signalling in neurons

doi: 10.1111/ejn.13766

Figure Lengend Snippet: Tctex1 interacts with MAP6 proteins. (A) Schematic representation of rat neuronal MAP6 proteins (MAP6‐N, MAP6‐E and MAP6d1) and the MAP6 fragments used for two‐hybrid screenings. Note that the N‐terminal (LNt), central (5R) and C‐terminal (LCt) fragments are complementary. Coloured modules: mauve, palmitoylable N‐terminal domain (Gory‐Faure et al ., ); light orange: MT‐stabilizing domains (Bosc et al ., ); dark grey: thermo‐sensitive MT‐binding domains (Delphin et al ., ); light grey: 28 C‐terminal repeats (Bosc et al ., ); light blue: consensus Tctex1‐binding sequence RRRIR (Mok et al ., ); black: AAAIA‐mutated corresponding sequence; hatched: MAP6d1‐specific aa with no homology to MAP6 proteins. The aa numbers are noted above. (B) Growth selection of yeasts containing the only baits LNt or LCt (top row) or a combination of the Tctex1 prey and either bait, either on histidine‐free medium (middle row) or visualization of protein interaction by staining of β‐galactosidase activity (bottom row). (C) Immunoprecipitation of protein complexes obtained from COS‐7 cells transfected with plasmids encoding Tctex1‐GFP and MAP6‐N, using sepharose beads coupled to either an anti‐GFP antibody or a mix of anti‐MAP6 antibodies (23N and 23C). Cell lysates (CL) and immunoprecipitated protein complexes (IP) were then analysed by SDS‐PAGE and Western blotting, using an anti‐GFP antibody (to evidence Tctex1) or an anti‐MAP6 antibody (175, to evidence MAP6). Control experiments were performed with no antibody coupled to the beads (no Ab). (D) Summary table of the two‐hybrid interactions assessed from growth on histidine‐free medium and staining of β‐galactosidase activity as described in (B). +++: very fast growth and strong staining; ++: medium growth and staining; +: slow growth and weak staining; ‐: no growth nor staining. (E) Schematic representation of MAP6‐N C‐termini (LCt, upper panel) used for immunoprecipitation with Tctex1‐myc (lower panel). Colour code is presented as in (A). Each immunoprecipitation was performed with an anti‐myc antibody and revealed with an immunoblot antibody against Tctex1 (Antibodies 2025 and 2026, see ) or an anti‐MAP6 (175) to evidence MAP6‐N C‐termini. MAP6‐related bands were quantified, background subtracted and normalized by that obtained with non‐mutated LCt (italic percentages). (F) Schematic representation of full‐length MAP6‐E or MAP6‐E mutants Δ1, ΔMn3 AAAIA or Δ1ΔMn3 AAAIA (lower panel) used for immunoprecipitation with Tctex1‐GFP (upper panel). Colour code is presented as in (A). Each immunoprecipitation was performed with the anti‐MAP6 23N antibody and immunorevealed by Western blotting against GFP or MAP6 (23N). Note that the many MAP6 bands detected after immunoprecipitation correspond to specific degradation products as there is no signal in the sham‐transfected condition. The contaminating IgG band, close to that of Tctex1‐GFP, is indicated. Tctex1‐related bands were quantified, background subtracted and normalized by that obtained with non‐mutated MAP6‐E (italic percentages). [Colour figure can be viewed at wileyonlinelibrary.com].

Article Snippet: Cell lysates were then incubated for 2.5 h at 4 °C with Sepharose beads (GE Healthcare, France) coupled with either protein G + rabbit anti‐GFP (Life Technologies, Gaithersburg, USA) or anti‐MAP6 antibodies [23N + 23C (Guillaud et al ., )] or protein A+mouse anti‐myc antibody (sc‐40, Santa Cruz Biotechnology Inc., Germany), before performing a 3‐min centrifugation at 10 000 g to collect the beads.

Techniques: Binding Assay, Sequencing, Selection, Staining, Activity Assay, Immunoprecipitation, Transfection, SDS Page, Western Blot