Ab11257

The anti-TEX12 (ab122455), anti-pericentrin (ab28144), anti-gamma tubulin (ab191114), anti-centrin 1 and 2 (ab11257) and anti-SYCE2 (ab107745) antibodies were obtained from Abcam and anti-FLAG (F1804) antibody was purchased from Sigma. Human TEX12 sequences, corresponding to amino acids 1–123 (full length) and 49–123 (core) and 45–123, 1–113, 1–91, 1–56 and 87–123, and SYCE2 were cloned into the pCMV-HA vector (Addgene Catalogue no 631604) for mammalian expression with an N-terminal FLAG-tag.

Cells were grown on slides placed in six-well culture plates at 70% confluence. They were fixed with cold methanol for 10 min and permeabilized with cold acetone for 1 min. The non-specific binding was blocked with PBS and 1% albumin for 1 h at room temperature. Primary antibodies anti-centrin 1 (ab11257, Abcam, 1:300) and anti-γ-tubulin (GTU-88, Sigma Aldrich, 1: 1000) were incubated at 4 °C overnight in a humid chamber. Secondary antibodies (ab175700, Abcam, 1:200 and ab150081, Abcam, 1: 500) were incubated for 1 h at room temperature. The DNA was stained with DAPI (Vectashield Mounting Medium with DAPI, Vector Laboratories, Burlingame, CA). The digital images were acquired using Zen lite software (Carl Zeiss, Gottingen, Germany) and AxioImager A2 microscope (Carl Zeiss, Gottingen, Germany) with Axiocam ICc5 camera and an αPlan-FLUAR 100×/1.45 oil objective.