Anti α tubulin clone dm1a

Flower buds were fixed for 45 min in ice-cold 4% (w/v) paraformaldehyde either in 1 × PBS (pH 7.4) or in 1 × microtubules stabilizing buffer (MTSB) buffer (50 mM PIPES, 5 mM MgSO₄ and 5 mM EGTA, pH 7.2) to preserve microtubules. All following steps were carried out with the same buffer used for fixation, that is, either PBS or MTSB. After washing in 1 × PBS/MTSB, meiotic chromosome spreads were prepared by squashing. Immunolabelling was performed as described56 (link). The following dilutions of primary antibodies were used: 1:100 of a rabbit anti-LnCENH3 (ref. 34 (link)), 1:200 of a monoclonal mouse anti-α-tubulin (clone DM 1A, Sigma), 1:250 of a rabbit anti-Zyp1 (ref. 42 (link)), 1:250 of a rabbit anti-Asy1 (ref. 57 (link)), 1:200 of a anti-H2A120phos (Abcam, ab111492), 1:100 of a rabbit anti-H3S28phos (Millipore, 07–145) and 1:300 of a monoclonal mouse anti-H3S10ph (Abcam, ab14955) antibody. A Cy3-conjugated anti-rabbit IgG (Dianova) and a fluorescein isothiocyanate-conjugated anti-mouse Alexa 488 antibody (Molecular Probes), each at a 1:400 dilution, were used as secondary antibodies.

Western blotting was performed as described previously52 (link). Briefly, protein lysates were prepared in RIPA buffer supplemented with complete protease inhibitor (Roche Applied Science). Proteins were then separated on 6–15% SDS-PAGE, transferred onto PVDF membranes, and probed with anti-CIBZ [amino acids 1184–1197 (EQKDDIKAFAENVL) of CIBZ]17 (link), anti-Oct3/4 (MAB1759, R&D Systems), anti-Sox2 (S1451, Sigma-Aldrich), anti-Nanog (AB5731, Millipore), anti-α-tubulin (clone DM 1A, Sigma-Aldrich), anti-Brachyury (sc-17743, Santa Cruz Biotechnology), anti-Nkx-2.5 (sc-8697, Santa Cruz Biotechnology), anti-Islet 1 (ab109517, Abcam), anti-cTnI (ab19615, Abcam), and anti-MHC (clone MF20, Developmental Studies Hybridoma Bank) antibodies. HRP-conjugated anti-mouse or anti-rabbit IgG (Cell Signaling) were used as secondary antibodies.

Transfected cells were plated in 60-mm culture dishes in serum containing medium. Then, cells were washed twice with PBS and cultured in serum-free medium for the indicated times.
Western blotting was done using standard procedures. The following antibodies were used: anti-phospho p42/p44 MAPK (ERK1/2) (Thr202/Tyr204); anti-p42/p44 MAPK (ERK1/2); anti-phospho AKT (Ser473); anti-AKT; anti-phospho STAT3 (Tyr705, clone 3E2); anti-STAT3 (Cell Signaling Technology, Danvers, MA,USA); anti-α-tubulin clone DM1A (Sigma-Aldrich, Milan, Italy). Densitometric analysis of the blots was performed using the ImageJ software.

The anti-CCDC74A/B rabbit antibody (TA344341, OriGene) was used for Western blotting (WB, 1:1000) and immunofluorescence (IF, 1:100). The other antibodies used for WB or IF are as follows: anti-acetylated tubulin (T7451, Sigma, RRID:AB_609894) (IF, 1:100), anti-Flag (clone M2, F3165, Sigma, RRID:AB_259529) (WB, 1:1000; IF, 1:100), anti-α-tubulin (clone DM1A, T9026, Sigma, RRID:AB_477593) (WB, 1:5000; IF, 1:500), anti-GAPDH (clone GAPDH-71.1, G9295, Sigma, RRID:AB_1078992) (WB, 1:1000), anti-green fluorescent protein (GFP; clone RQ2, D153-3, MBL, RRID:AB_591817) (WB, 1:100), anti-GST (sc-33613, Santa Cruz Biotechnology, RRID:AB_647588) (WB, 1:1000), anti-TACC3 (sc-376883, Santa Cruz) (IF, 1:200), anti-TPX2 (11741-1-AP, Proteintech, RRID:AB_2208895) (WB, 1:200; IF, 1:50), anti-γ-tubulin (T3559 and T6557, Sigma, RRID:AB_477575 and RRID:AB_477584) (IF, 1:200), and anti-Pericentrin (ab4448, Abcam, RRID:AB_304461) (IF, 1:1000). We used Alexa Fluor 488/568-conjugated goat anti-mouse/rabbit IgG (Alexa Fluor series, Molecular Probes) (IF, 1:200) as secondary antibodies for immunofluorescence and HRP-conjugated goat anti-mouse/rabbit IgG (Jackson ImmunoResearch) (WB, 1:5000) as secondary antibodies for Western blotting.

Western blotting was performed as previously described (23 (link)). In brief, whole cell lysates were harvested from cultured cell lines in Phospho-Safe extraction reagent (EMD Millipore) and protein was quantified using the BCA assay (Thermo Scientific). Lysates were separated by SDS-PAGE on 10-12% polyacrylamide gels and transferred to polyvinylidene difluoride membranes (EMD Millipore). Except of anti-α-Tubulin (clone DM1A, Sigma), phospho-PRAS40 (Thr246) (polyclonal, Thermo Scientific) and anti-c-MYC (clone Y69, Abcam), all other antibodies were purchased from Cell Signaling: anti-phospho-AKT (Ser473) (clone D9E), anti-phospho-AKT (Thr308) (clone C31E5E), anti-phospho-S6 ribosomal protein (Ser235/236) (Cat# 2211) and anti-AKT (Cat# 9272).