Arellano & Briseño et al Supporting Information
SUPPORTING MATERIAL AND METHODS
Strain list
N2, AX204, npr-1(ad609) X, AX51, maco-1(db1) I; npr-1(ad609) X, AX59, maco-1(db9) I; npr-1(ad609) X,
AX2208, maco-1(db129) I; npr-1(ad609) X, AX2193, maco-1(db1) I, AX2255, maco-1(db9) I, AX2206,
maco-1(db129) I, AX2197, lin-15(n765ts) X ; dbEx450[yfp::pis, lin-15(+)], AX2203, lin-15(n765ts) X;
dbEx451[yfp::MANS, lin-15(+)], AX2198, lin-15(n765ts) X ; dbEx452[yfp::TRAM, lin-15(+)], AX2204,
lin-15(n765ts) X ; dbEx453[yfp::EMERIN ,lin-15(+)], CZ900, syd-2(ju37) X , CB193, unc-29(e193) I ,
MT150, egl-3(n150) V, AX2199, juIs1[punc-25::snb-1::gfp], AX2372, hpls3 [punc-25::syd-2::gfp],
AX2372, hpls61[punc-25::unc-10::gfp], AX2201, maco-1(db9) I; juIs1[punc-25::snb-1::gfp], AX2368,
maco-1(db9) I; hpIs3 [punc-25::syd-2::gfp], AX2369, maco-1(db9) I; hpls61[punc-25::unc-10::gfp],
AX2370, egl-19 ; npr-1(ad609) X, AX2371, nca-1; nca-2 ; npr-1(ad609) X, AX1339, npr-1(ad609) X;
dbIs[pgcy-36::snb-1::yfp], AX218, osm-9(n1601) IV; npr-1(ad609) X, AX1059, tax-4(ky89) III; npr1(ad609) X, AX2194, maco-1(db9) I; npr-1(ad609) lin-15(n765ts) X; dbEx454[pmaco-1::maco-1], AX2195,
maco-1(db129); npr-1(ad609) lin-15(n765ts) X; dbEx455[FosWRM0640bE08], AX1864, npr-1 (ad609) lin15(n765ts) dbEx[Pgcy-32::YC3.60; lin15+], AX2249, maco-1 (db9) ; npr-1 (ad609) lin-15(n765ts)
dbEx[Pgcy-32::YC3.60;lin15+], AX2251, egl-19(ad1006) ; npr-1 (ad609) lin-15(n765ts) dbEx[Pgcy32::YC3.60; lin15+], AX2395, nca-1(gk9); nca-2(gk5); npr-1(ad609) lin-15(n765ts) dbEx[Pgcy32::YC3.60; lin15+].
Immunostainings
Fixing formulation: 2% (w/v) PFA, 80 mM KCl, 20mM NaCl, 15mM EGTA, 15mM PIPES, 0.5mM
Spermidine, 20% (v/v) Methanol, pH 7.4. Fix wash solution: 100 mM Tris HCL, 1% (v/v) Triton X-100,
1mM EDTA, pH 7.4. Permeabilising solution: 2% (v/v) -mercaptoethanol, 100mM Tris HCL, 1% (v/v)
Triton X-100, 1mM EDTA, pH 7.4. AbB buffer: 1x PBS, 0.2% (v/v) BSA Fraction-V, 0.5% (v/v) Triton X100, 1mM EDTA. AbA buffer: 1x PBS, 2% (w/v) BSA Fraction-V, 0.5% (v/v) Triton X-100, 1mM EDTA.
Antibody preparation
DNA encoding the C-terminal domain of MACO-1 (construct named C2E9) was amplified from the maco-1
Arellano & Briseño et al Supporting Information
cDNA and cloned in a His tag containing vector. The construct was expressed in E. coli and the sonicated
crude bacterial lysates were passed through a Ni-NTA (Qiagen) column pre-equilibrated with Ni-Lysis
Buffer. Fractions were eluted and pooled and dialysed in the presence of 2 mgs TEV protease, and
subsequently passed through a second Ni-NTA column. The flow-through was concentrated and run in a gel
filtration Superdex-75 column (Pharmacia – Pfizer) previously equilibrated with 1x PBS plus 100 mM
NaCl. The eluted fractions were pooled, concentrated, and the final concentration was determined by
Abs280nm using a predicted extinction coefficient of 280nm =11,595 M-1 cm-1. Aliquots of the purified
domain were sent to Eurogentec (Brussels, Belgium) for the production of rabbit polyclonal antibodies. The
purified C-terminal domain (C2E9) was immobilised in HiTrap NHS-activated (1mL) columns (Amersham
Biosciences, UK) following the manufacturers protocol. 50 mL of the final bleed of the immunogenised
serum were diluted in 2x PBS with 0.1% NaN3 and filtered through a 0.20 m membrane. The diluted serum
was circulated overnight at 4 °C through the HiTrap-C2E9 coupled column. The loaded columns were
thoroughly washed (>150 column volume) with Wash Buffer and 1x PBS (> 100 column volume). The
columns were eluted with 0.1M glycine pH 2.6 in ~1mL fractions, with an immediate pH neutralization with
200 l of 2 M Tris-HCl pH 7.4. The absorbance at 280nm was measured for all fractions, and those with an
Abs280nm > 0.1 were pooled and sequentially dialyzed against 1x PBS and then 1x PBS, 60% (v/v)
glycerol. Aliquots were prepared, frozen in liquid nitrogen, and stored at –80 °C until further used. Ni-Lysis
Buffer: 50mM Potassium phosphate, 300mM NaCl, 10mM Imidazole, pH 8.0, Complete EDTA-free
protease inhibitor (Roche Diagnostics, Germany). Ni-Elution Buffer: 50 mM Potassium phosphate, 300 mM
NaCl, 250 mM Imidazole, pH = 8.0. Affinity washing solution: 1x PBS, 0.5 M NaCl, 0.1% (v/v) Triton X100.
Co-localisation microscopy
Images were acquired using an Axio Imager.Z1 microscope with LSM510 scanning module (Carl Zeiss).
Images were acquired at between 19 and 22 °C. The objective used was a Plan-Apochromat 63x/1.4 Oil DIC.
Each image was scanned four times. Filters used were LP560, BP 420-480, BP 505-530 for channels 1, 2 and
3 respectively.
Arellano & Briseño et al Supporting Information
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