αγ tubulin

Polyclonal αpY397-FAK (Abcam), monoclonal αFAK (EMD Millipore), polyclonal αpS298-MEK (Cell Signaling), monoclonal αMEK (GeneTex), monoclonal αphospho-ERK1/2 (pT202/Y204) and polyclonal αERK1/2 (Cell Signaling), monoclonal αmyc (9E10, Santa Cruz Biotechnology), and αγ-tubulin (Sigma-Aldrich) were used for immunoblotting. Na₃VO₄ was purchased from Sigma. siRNA and primers for PTP-PEST were purchased from Santa Cruz Biotechnology. Control nontargeting siRNA was purchased from Cell Signaling. Human PRL was purchased from the National Hormone and Peptide Program (Dr. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases).

Samples for immunoblots of mammalian proteins were prepared by 3 × wash with cold 1 × PBS and kept at −80 °C until 2 × Laemmli buffer was added to make whole cell lysates. Total cell extracts for immunoblots of S. pombe proteins were made by TCA protein extraction^{53 (link)}. Antibodies for immunoblots were α-γtubulin (1:10000, T6557, Sigma-Aldrich), α-CHK1-P345 (1:1000, 2348, Cell Signaling Technology), α-CHK1 (1:200, DCS310.1, Santa Cruz), α−4EBP1 (1:2000, Cell Signaling Technology) α-phosphoAkt substrates (1:1000, 23C8D2, Cell Signaling Technologies), α-PSTAIRE, recognizing a motif in cdc2 (1:2000, Santa Cruz Biotechnology sc-53) and anti-peroxidase PAP1, against the TAP-tap (1:1000, Sigma P1291). Appropriate ECL and ECF kits were used for detection.

Primary antibodies used for immunofluorescent markers of the centrosome were α-GM130 (BD 610822, 1:1,000 dilution [dil.]) and α-γ-tubulin (Sigma Aldrich T5326-25UL, 1:1,000). The secondary used for both stains was α-Ms-Alexa-488 (Invitrogen A28175, 1:1,000). Tissue fixation and staining was carried out using standard protocols using cold methanol (52 ). Immunofluorescent samples were imaged using confocal microscopy (see below). Antibodies used for Western blotting and immunofluorescence were α-β-catenin (Cell Signaling, #2698S, 1:1,000) and α-β-actin (Sigma, A3853, 1:1,000). Secondary antibodies used were α-Gt-680RD and α-Ms-800CW (Licor 926-6807 and 926-32212, respectively, both 1:10,000 dil.). Standard immunoblot procedures were used (53 ).