γ tubulin

The indicated antibodies against the following proteins/peptides were used for Western blot analyses: Flag (F3165; Sigma-Aldrich), HA (MMS-101P; Covance), Myc (sc-40; Santa Cruz Biotechnology), GFP (sc-9996; Santa Cruz Biotechnology), WWP1 (H00011059-M01; Novus Biologicals), WWP2 (A302-935A; Bethyl Laboratories), AMOTL2 (sc-82501; Santa Cruz Biotechnology), USP9X (Abnova, H00008239-M01), CRB3 (NBP1-81185; Novus Biologicals), PALS1 (Santa Cruz, sc-365411), LATS1 (A300-477A; Bethyl Laboratories), LATS2 (#5888; Cell Signaling Technology), p-LATS1 (#8654; Cell Signaling Technology), YAP p-S127 (#4911; Cell Signaling Technology), YAP (H00010413-M01; Novus Biologicals), TAZ (#4883S; Cell Signaling Technology), CTGF (sc-14939; Santa Cruz Biotechnology), CYR61 (sc-13100; Santa Cruz Biotechnology), ubiquitin (550944; BD Biosciences), γ-tubulin (sc-7396; Santa Cruz Biotechnology), occludin (71-1500; Invitrogen), lamin B (sc-6217; Santa Cruz Biotechnology), and GAPDH (ab125247; Abcam); rabbit anti-AMOTL2 p-S159 was a kind gift from Dr Wanjin Hong (National University of Singapore). Antibodies used for immunofluorescence analyses were: YAP (H00010413-M01; same as for Western blot; Novus Biologicals), GFP (ab13970; Abcam), and BrdU (555627; BD Biosciences). The antibodies used for immunoprecipitations were the same as those used for Western blot analyses.

Antibodies against the following proteins (for immunostaining, western blotting, or IPs) were used in this study as described previously: SMG6^{74 (link)}, UPF1^{75 (link)}, and CTIF^{68 (link)}.
The purchased antibodies against the following proteins are listed in the format “protein name (catalog number, supplier)”: FLAG (DYKDDDDK; 14793, Cell Signaling Technology or A8592, Sigma-Aldrich), Myc (9E10; OP10L, Calbiochem or 2272, Cell Signaling Technology), FTO (ab124892, Abcam), GFP (sc-9996, Santa Cruz Biotechnology), DCTN1 (p150glued; 610474, BD Biosciences), eEF1A1 (CBP-KK1; EF1α; 05-235, Merck Millipore), YTHDF1 (17479-1-AP, Proteintech), YTHDF2 (24744-1-AP, Proteintech), YTHDF3 (sc-377119, Santa Cruz Biotechnology), METTL3 (15073-1-AP, Proteintech), METTL14 (HPA038002, Sigma-Aldrich), p-(S/T)Q ATM/ATR substrate (2851, Cell Signaling Technology), m⁶A (#202003, Synaptic Systems), m¹A (D345-3, MBL), puromycin (12D10; MABE343, Merck Millipore), β-actin (A5441, Sigma-Aldrich), GAPDH (LF-PA0212, AbFrontier), α-tubulin (sc-53030, Santa Cruz Biotechnology), γ-tubulin (sc-17788, Santa Cruz Biotechnology), dynein (sc-9115, Santa Cruz Biotechnology), G3BP1 (13057-2-AP, Proteintech), IMPβ (A301-803A-1, Bethyl Laboratories), Alexa Fluor 488 goat α-mouse IgG (A-11017, Invitrogen), and rhodamine-conjugated goat α-rabbit IgG (31670, Invitrogen).

Cells were centrifuged, washed in ice-cold PBS buffer, and then lysed in RIPA lysis buffer. The amount of protein was quantified using a protein assay kit (Bio-Rad, Korea). Each sample was subjected to SDS-PAGE and transferred to an Immobilon Transfer Membrane (Millipore, Cat#IPVH00010). The filter was incubated with each corresponding antibody, and immunodetection was carried out using the PowerOpti-ECL Western blotting detection reagent (Bio-Rad). The antibodies used in this study were purchased as follows: Bax (Santa Cruz, sc-20067), cyclophilin A (CypA)(Enzo Life Sciences, BML-SA296), HIF-1α (Cell Signaling, Cat#3716S), HMBG1 (Enzo Life Sciences, ALX-210-964), LC3 (Enzo Life Sciences, ALX-803-082), p62/SQSTM1(Cell Signaling, Cat#5114), RIP3 (Santa Cruz, sc-47368), TRIP-Br1 (Enzo Life Sciences, ALX-804-645), XIAP (Cell Signaling, Cat# 2042) and γ-tubulin (Santa Cruz, sc-7396).

For staining of cultured cells, cells were grown on coverslips, fixed with 4% paraformaldehyde for 10 min and permeabilized with PBS, 0.1% Triton X-100 for 15 min, or fixed and permeabilized with 100% methanol for 10 min. Blocking was carried out with PBS, 0.1% Tween 20, 0.5% BSA (PBT/BSA), followed by antibody incubation for 60 min. Primary antibodies and dilutions (in PBT/BSA) were: E-cadherin (CDH1) 1:200 (BD Biosciences, 610181), β-catenin 1:400 (BD Biosciences, 610153), 5hmC 1:2,000 (Active Motif, 39769), TET1 1:750 (GeneTex, GTX125888), TET2 1:100 (Abcam, ab124297), CDX2 1:400 (BioGenex, MU392-UC), α-tubulin 1:1,000 (Abcam, ab6160), and γ-tubulin 1:250 (Santa Cruz, sc7396). Primary antibodies were detected with the appropriate secondary Alexa Fluor 488 or 568 (Thermo Fisher Scientific) antibodies. Nuclei were counter-stained with DAPI. Photographs were taken with an Olympus BX61 epifluorescence microscope or a Zeiss LSM 780 confocal microscope.
Nuclear morphology (shape) was analyzed in ImageJ. In brief, after application of a background filter separate images were created of nuclei and donut holes. Their segmentation allowed for the identification of donut-shaped nuclei by the presence of a hole. Data were processed in Excel.