Sc 50164

Cells were grown on 12-mm round coverslips to 50–70% confluence at the time of fixation. Cells were fixed either in paraformaldehyde or methanol, depending upon manufacturers’ recommendations for the primary antibodies. The blocking reagent used was 4% BSA (bovine serum albumin) in PBS, and the primary antibodies used were OFD1 (1:100, Origene), ɣ-Tubulin (1:1000; T5326, Sigma-Aldrich), α-tubulin (1:2000, ab7291, Abcam), PCM1 (1:100, sc-50164, SantaCruz Biotechnology, Dallas, Texas), GM-130 (1:250, 610822, BD Biosciences, San Jose, California), TGN-46 (1:200, GTX74290, Genetex, Irvine, California), or KDEL (1:100, ab12223, Abcam). Alexa Fluor conjugated secondary antibodies used were Alexa488 Donkey anti-Rabbit, Alexa555 Donkey anti-Mouse, and Alexa647 Donkey anti-Rabbit (ThermoFisher Scientific). Where indicated, DNA was counterstained with Hoechst 33342 (ThermoFisher Scientific) and coverslips were rinsed with PBS. The mounting medium was ProLong Gold antifade mounting medium (ThermoFisher Scientific) and the slides were imaged using a 20× or 40× objective on a Zeiss LSM 700 confocal laser-scanning microscope (Carl Zeiss Microscopy, GmbH, Jena, Germany).

GST pull-down was performed with Glutathione Sepharose beads (GE healthcare, Chalffint, UK) and carried out in low-salt EBC lysis buffer (150 mM NaCl; 50 mM Tris, pH 7.5; 1 mM EDTA; 0.5% NP40). Antibodies included in this study: CEP131 (A301-425A, Bethyl, WB 1:1000; ab84864, Abcam, Cambridge, UK, IF 1:300), PCM1 (sc-50164, Santa Cruz, IF 1:200), SSX2IP (HPA027306, Sigma-Aldrich, WB 1:1000), MK2 (3042S, Cell Signalling, Danvers, MA, USA, WB 1:1000), HSP27 pS82 (9709, Cell Signalling, WB 1:5000), GST (G7781, Sigma-Aldrich, WB 1:1000), Pericentrin (ab4448, Abcam, IF 1:1000), p38 MAPK (9212S, Cell Signalling, WB 1:1000), p38 MAPK phospho-Thr180/Tyr182 (9216S, Cell Signalling, WB 1:1000), γ-tubulin (T5326, Sigma-Aldrich, IF 1:250), p150 (610473, BD Biosciences, WB 1:10,000), NOGO-B (previously described by Rousseau et al. [66 (link)] WB 1:1000), SRF phospho-Ser103 (4261, Cell Signalling, WB 1:1000), and α-tubulin (T9026, Sigma-Aldrich, IF 1:500).