Rabbit anti pericentrin

Antibodies monoclonal against Ac-Tub and α-SMA were from Sigma-Aldrich (Saint Quentin Fallavier, France), rabbit against Kif-3A was from Abcam (Cambridge, UK) rabbit anti-AKT, phospho-Akt (thr 308), Smad, phospho-Smad, ERK, phospho-ERK were from Cell Signaling (Ozyme, St Quentin en Yvelines, France), monoclonal anti ARL13B (sc-515784) was from Santa Cruz (Ozyme, St Quentin en Yvelines, France), rabbit anti pericentrin was from Bethyl (Euromedex, Souffleweyrsheim, France). Antibody against mouse coupled to Alexa Fluor 488 and antibody against rabbit coupled to Alexa fluor 647 were from Life Technologies (Saint Aubin, France). Antibodies against CD56 APC and CD140a PE were purchased from BD-Biosciences (Le Pont de Claix, France). HPI-4 was from Sigma-Aldrich. Shh-conditioned medium was obtained from an HEK 293 cell line stably transfected with Shh-N expression vector (ATCC #CRL-2782).

Cells were fixed in either 4% PFA for 20 min, in ice-cold methanol for 10 min (for γ-tubulin and centrin antibodies) or for 10 min in 1× PTEMF extraction buffer (20 mM PIPES, pH 6.8, 10 mM EGTA, 1 mM MgCl 2, 0.2% Triton X-100 and 4% PFA) prepared fresh from 4× stock solution (for all the mitotic checkpoint antibodies). Cells were permeabilized by 2×3 min wash in 0.1% Triton-X-PBS (PBS-TX), followed by blocking in PBS-TX containing 1% BSA for 30 min at room temperature, before being processed for immunofluorescence. Primary antibodies were diluted in 1% BSA in 1× PBS-TX in a 37°C incubator for 1 hr. Primary antibody dilutions were as follows: mouse anti-α-tubulin (DM1α, Sigma, 1∶1000), rabbit anti-γ-tubulin (Sigma, 1∶1000), rabbit anti-pericentrin (Bethyl, 1∶300), mouse anti-centrin-3 (Abnova, 1∶500), rabbit anti-TPX2 (Bethyl, 1∶100), mouse anti-Hec1 (GeneTex, 1∶100), human anti-centromere antibody (ACA, 1∶2000), mouse anti-BubR1 (Millipore, 1∶50), mouse anti-Mad1 (a kind gift from Dr. Andrea Musacchio, 1∶300), sheep anti-Mad2 (a kind gift from Dr. Steven Taylor's Laboratory, 1∶200), rabbit anti-Zwint-1 (Bethyl, 1∶100). Secondary antibodies conjugated to FITC, TRITC or Cy-5 (all from Jackson Laboratories) were chosen as appropriate and used as recommended by the supplier. DNA was counterstained with DAPI.