α tubulin dm1a

To perform immunostaining, the following antibodies were used at 1:2000–3000 dilutions: α-Tubulin (DM1a; Sigma, St-Louis, MO), α-GFP: IgG₁κ (Roche, Indianapolis, IN), α-ZYG-1 (Stubenvoll et al. 2016 (link)), α-TBG-1(Stubenvoll et al. 2016 (link)), α-SAS-4 (Song et al. 2008 (link)), α-SAS-5 (Medley et al. 2017 (link)), and Alexa Fluor 488 and 561 (Invitrogen) as secondary antibodies. Confocal microscopy was performed as described (Stubenvoll et al. 2016 (link)) using a Nikon Eclipse Ti-U microscope equipped with a Plan Apo 60 × 1.4 NA lens, a Spinning Disk Confocal (CSU X1) and a Photometrics Evolve 512 camera. Images were acquired using MetaMorph software (Molecular Devices, Sunnyvale, CA). MetaMorph was used to draw and quantify regions of fluorescence intensity and Adobe Photoshop CS6 was used for image processing. To quantify centrosomal SAS-5 signals, the average intensity within an 8-pixel (1 pixel = 0.151 μm) diameter region was measured within an area centered on the centrosome and the focal plane with the highest average intensity was recorded. Centrosomal TBG-1 (γ-tubulin) levels were quantified in the same manner, except that a 25-pixel diameter region was used. For both SAS-5 and TBG-1 quantification, the average fluorescence intensity within a 25-pixel diameter region drawn outside of the embryo was used for background subtraction.

For western blotting, samples were sonicated for 5 min and boiled in a solution of 2× Laemmli Sample Buffer and 10% β-mercaptoethanol before being fractionated on a 4–12% NuPAGE Bis-Tris gel (Invitrogen). The iBlot Gel Transfer system (Invitrogen) was then used to transfer samples to a nitrocellulose membrane. The following antibodies were used at 1:3000–10,000 dilutions: α-Tubulin: α-Tubulin (DM1a; Sigma), α-GFP: IgG₁κ (Roche), α-SAS-5 (Song et al. 2011 (link)), and α-TBG-1 (Stubenvoll et al. 2016 (link)). IRDye secondary antibodies (LI-COR Biosciences, Lincoln, NE) were used at a 1:10,000 dilution. Blots were imaged using the Odyssey infrared scanner (LI-COR Biosciences), and analyzed using Image Studio software (LI-COR Biosciences).

The primary antibodies for immunostaining were GT335 (Gift from B. Edde and Dr. C. Janke, 1:2000), Neurofilament M (RMO14.9; Cell Signaling Technology, 1:25), and Map2 (ab5622; Merck Millipore, 1:1000). The secondary antibodies were Alexa fluor 488 (Thermo Fisher Scientific, 1:1000) and Alexa fluor 568 (Thermo Fisher Scientific, 1:1000). The antibodies used for immunoblot were TTLL1 (Ikegami et al., 2010, 1:1000), TTLL7 (Ikegami et al., 2006, 1:1000), GT335 (1:20,000), α-tubulin (DM1A; Sigma-Aldrich, 1:50,000), β-tubulin (TUB2.1; Sigma-Aldrich, 1:2000), tyrosinated tubulin (1A2; Sigma-Aldrich, 1:2000), acetylated tubulin (6-11B-1; Sigma-Aldrich, 1:20,000), neurofilament 200 (NE14; Sigma-Aldrich, 1:500), neurofilament 160 (NN18; Sigma-Aldrich, 1:250), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 6C5; Sigma-Aldrich, 1:5000), CLIP170 (Sigma-Aldrich, 1:500), Map1A (HM-1; Sigma-Aldrich, 1:1000), Map1B (Sigma-Aldrich), 1:1000; Map2 (HM-2; Sigma-Aldrich), 1:1000; Tau-1 (MAB3420; Merck Millipore, 1:10,000), dynein (74.1; Merck Millipore, 1:1000), KIF1A (no. 16; BD Biosciences, 1:500), KIF5 (H2; Merck Millipore, 1:500), and KIF17 (Sigma-Aldrich, 1:1000). The antibodies used for 2D electrophoresis were GT335 (1:20,000), α-tubulin (DM1A; 1:4000), β-tubulin (TUB2.1; 1:4000).

Antibodies reactive to Bcl-rambo (Rocky-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (7H8.2C12; BD Biosciences, San Jose, CA, USA), FLAG (1E6, Wako Pure Chemical Industries, Osaka, Japan), HSP60 (insect) (Enzo Life Science, Farmingdale, NY, USA), α-tubulin (DM1A; Sigma-Aldrich, St. Louis, MI, USA), and VSV-G (P5D4; Santa Cruz Biotechnology) were commercially obtained. Antibodies reactive to Cut (2B10), Discs large (4F3), Elav (7E8A10), and Prospero (MR1A) were obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA, USA). Secondary antibodies conjugated to horseradish peroxidase (HRP) were purchased from Jackson ImmunoResearch (West Grove, PA, USA).

Antibodies were purchased as follows: BIM (C34C5), BCL-XL (54H6), CREB (48H2) and cleaved caspase-3 (5A1E) from Cell Signaling Technology (Danvers, MA, USA); NOXA (114C307.1) from Thermo Fisher Scientific (Waltham, MA, USA); ATF-3 (C-19) and MCL-1 (S-19) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); KLF4 (56CT5.1.6) from Bioss, Inc. (Woburn, MA, USA); α-tubulin (DM1A) and β-actin (AC-74) from Sigma-Aldrich (Tokyo, Japan). APTO-253 was purchased from MedChemExpress, LLC (Monmouth Junction, NJ, USA). Q-VD-OPh, Doxorubicin, etoposide, SP600125, SB203580, PD184352, and U0126 were purchased from Calbiochem (San Diego, CA, USA). Hydroxychloroquine (HCQ), Necrostatin-1 and Ferrostatin-1 were purchased from Cayman Chemical (Ann Arbor, MI, USA).

For immunoprecipitation (IP), cells were lysed in CHAPS lysis buffer (40 mM Hepes pH7.4, 120 mM NaCl, 2 mM EDTA, 0.3% CHAPS) containing mix of protease inhibitors (Boehringer), plus 5 mM NaF, 10 mM glycerophosphate and 1 mM Na₃VO₄. For IP lysates were pre-incubated with protein G-Agarose (Pierce) and then with the indicated antibody, under gentle rocking at 4 °C overnight. For Western blot (Wb) cells were lysed in RIPA buffer. Membranes were developed using the enhanced chemiluminescence (ECL Amersham) by chemiluminescence imaging system, Alliance 2.7 (UVITEC Cambridge) and quantified by the software Alliance V_1607. Primary antibodies used: MDM4 BL1258 (Bethyl laboratory), MDM4 C82 (Sigma), MDM4 8C6 (Millipore) p53 FL393 (Santa Cruz), α-tubulin DM1A (Sigma), actin C-40 (Sigma), mTOR (Santa Cruz), mTOR (Cell Signaling), anti-FLAG M2 affinity gel (Sigma), phosphoSer473-AKT (Cell Signaling), phosphor-Thr389-S6K (Cell Signaling), Akt (Cell Signaling), S6K1 (Santa Cruz), Raptor (Cell Signaling), Raptor (Santa Cruz).
Fractionation of lysates into heavy membrane and light membrane/cytosolic fractions was performed according to Menon et al. 2014.