Ab6160

Protein lysates and western blots were prepared according to routine protocols. Primary antibodies used were: cyclin D1 1:200 (Merck Millipore, cc-12), cyclin B1 1:500 (Neomarker, Thermo Scientific, MS-868), AURKB 1:1,000 (Cell Signaling Technology, 3094), P21 (CDKN1A) 1:500 (BD Biosciences, 556431), TET2 1:1,000 (Abcam, ab124297), tubulin 1:2,500 (Abcam, ab6160), actin 1:2,000 (Santa Cruz, sc7210), and HSP90 1:5,000 (BD Biosciences, 610418). For all western blot data, n specifies biological replicates.

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.

Primary antibodies used in this study were as follows: anti-BRAT1 (IF 1:500, WB 1:50,000; Abcam, ab181855), anti-INTS11 (WB 1:1000; Novus Biologicals, NB100-60638), anti-INTS11 (IF 1:500; Novus Biologicals, NBP3-03680), anti-INTS9 (WB 1:1000; Cell Signalling, 13945), anti-INTS4 (WB 1:1000; Abcam, ab75253), anti-INTS3 (WB 1:1000; Bethyl, A302-050A), anti-INTS1 (WB 1:1000; Bethyl, A300-361A), anti-β-actin (WB 1:5000; Protein Tech, 66009), anti-α-tubulin (WB 1:8000; Abcam, ab6160), anti-Lamin B (WB 1:500; Santa Cruz, sc-6216), anti-Coilin (IF 1:250, WB 1:1000; Santa Cruz, sc-32860), anti-B23 (IF 1:250; Santa Cruz, sc-271737) and anti-FLAG (IF 1:250, WB 1:500; Sigma, F1804). Secondary antibodies employed for western blotting were HRP-conjugated goat anti-rabbit (1:10,000; Bio-Rad, 170-6515), goat anti-mouse (1:10,000; Bio-Rad, 170-6516), rabbit anti-rat (1:10,000; Abcam, ab6734), for western blotting after immunoprecipitation HRP-conjugated light chain specific mouse anti-rabbit (1:10,000; Jackson Immunoresearch, 211-032-171) and for indirect immunofluorescence were goat anti-rabbit Alexa 488 (1:10,000; Invitrogen, A-11008) and donkey anti-mouse Alexa 647 (1:10,000; Invitrogen, A-31571).