Anti ha

Anti-WWP2, anti-PPM1G, anti-HA (all from Bethyl Laboratories), anti-WWP1 (Abnova), antiubiquitin (anti-Ub) (Millipore), anti-MBP (New England Bio Labs), anti-Myc clone 9E10 (Santa Cruz Biotechnology), anti-Flag, anti-GST, antiactin, and antitubulin (Sigma) antibodies were used in this study.

FANCL-deficient DT40 cells (fancl^−/−) and fancl^−/− complemented with TAP-tagged wild-type FANCL (TAP-FANCL) were described recently (27 (link)). Point mutations L7A, D78A, D78R, L79A, and V80A were generated by site-directed muagenesis (QuickChange II XL site-directed mutagenesis kit; Stratagene). DT40 cell transfections, DT40 subfractionation, Superpose 6 gel size chromatography, and FANCD2 immunoblot analyses were described previously (27 (link)). To assess TAP-FANCL interaction with ubiquitin, fancl^−/− cells expressing either TAP-FANCL or TAP-FANCL (L7A, D78R, L79A) were lysed in TAP-buffer (50 mm Tris, pH 8.0, 200 mm NaCl, 0.5 mm EDTA, 0.1% Nonidet P-40, and protease inhibitor mixture (Roche Molecular Biochemicals)). TAP-tagged FANCL variants were immunoprecipitated from 2 mg lysates with 50 μl of IgG-coupled Sepharose (GE Healthcare) and washed extensively with Ub-binding buffer (UbBB) (50 mm Tris, pH 7.4, 150 mm NaCl, 0.1% Nonidet P-40, 2 mg/ml BAS). TAP-FANCL Sepharose beads were homogenized in UbBB and incubated with 10 mg/ml HA-tagged wild type or I44A-mutated ubiquitin for 2 h at 4 °C. Beads were washed with wash buffer (50 mm Tris, pH 7.4, 250 mm NaCl, 0.2% Nonidet P-40). Bound ubiquitin was analyzed by immunoblotting with anti-HA (Bethyl Laboratories).

The following primary antibodies were used: anti-HA (chicken, A190-106A; Bethyl Laboratories), anti-actin (rabbit, A2066; Sigma), anti-myc (mouse, 9E10 clone; Juan Bonifacino, NIH), anti-myc (rabbit, 2278; Cell Signaling), anti-RhoA (mouse, ARH04; Cytoskeleton), anti-RhoA (mouse, SAB1400017; Sigma), anti-FLAG (mouse, F1804; Sigma), anti-FLAG (rabbit, 600-401-383; Rockland Immunochemical), anti-ARF1 (mouse, sc-53168; SCBT), anti-α-tubulin (mouse, T5168; Sigma), anti-acetylated α-tubulin (mouse, T6793; Sigma), anti-detyrosinated α-tubulin (rabbit, 48389; Abcam), ActiStain-488 (PHDG1; Cytoskeleton), Actistain-555 (PHDH1-A; Cytoskeleton), anti-heat shock protein 70 (HSP70) (chicken, SPC-178D; StressMarq), anti-InaC (mouse; T. Hackstadt), anti-IncA (rabbit; T. Hackstadt), and anti-MOMP (goat, 1621; ViroStat). The following secondary reagents were used: Hoechst dye (H1399) and goat and donkey anti-mouse, anti-goat, anti-rabbit, and anti-chicken (IgY) IgG Alexa Fluor 488-, 555-, or 647-conjugated secondary antibodies (Invitrogen). Donkey anti-chicken, anti-mouse, or anti-rabbit IgG and IgY horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Invitrogen.

Tissue samples were homogenate in ice-cold sample buffer (8M urea, 4% CHAPS, 40 mM Tris-base, 65 mM DTT containing a protease inhibitor cocktail) and after 30 minutes incubation on ice, samples were centrifuged at 13000 x g at 4°C. The surnatants were collected in new tubes and protein content was assayed by the Bradford dye-binding method (BioRad Protein assay).
Cells were plated in 100-mm culture dishes at a density of 5×10⁵ cells/ml. After treatments, cells were lysed and extracted as previously described [57 (link)].
For immunoblotting, the following primary antibodies were used: p53 specific DO-1 (Santa Cruz, California, USA 1:300), p53-Ser15P (Santa Cruz, California, USA 1:100), p53-Ser20P (Santa Cruz, California, USA 1:100), MDM2 specific 2A10 (Calbiochem 1:400), p21 specific C-19 (Santa Cruz, 1:200), TRIM8 specific C-20 (Santa Cruz, California, USA 1:200), Anti-HA (Bethyl Laboratories, 1:1000), Anti-Actin Ab-1 antibodies kit (Calbiochem, 1:2000). Bound primary antibodies were visualized using Lumi-Light Western Blotting Substrate (Roche™) on a UVITEC Cambridge Camera.

Anti-PHLPP1 (A300-660A from Bethyl Laboratories and 07-1341 from Millipore), anti-SGT1 (A302-944A from Bethyl Laboratories and Ab30931 from Abcam), anti-RNF41 (A300-048A from Bethyl Laboratories), anti-CENP-A (Ab13939 from Abcam), anti-CENP-E (Ab5093 from Abcam), anti-HEC1 (Ab3613 from Abcam) anti-Myc, clone 9E10 (SC-40 from Santa Cruz Biotechnology), anti-histone H3 (05928 from Millipore), anti-γ-tubulin (T5326 from Sigma), anti-α-tubulin (T6074 from Sigma), anti-FLAG (F3165 from Sigma), anti-actin (A5441 from Sigma), anti-HA (A190-108A from Bethyl Laboratories), and anti-ubiquitin (05-944 from Millipore) antibodies were used in this study.

HeLa cells were purchased from ATCC. The cell lines were validated using STR profiling and free from mycoplasma contamination for all experiments. Antibodies: anti-β-actin (Sigma, A5441); anti-HA (Bethyl Laboratories, A190-108A); anti-FLAG (Sigma, F1084); anti-Myc (PTM Bio, #PTM-5390); and anti-USP16 and anti-USP16-pS552 antibodies were described before (27 (link)); anti-OGT (Abcam, AB96718);RL2 (Abcam AB2739); anti-Histone H2A (CST #2578); anti-uH2A (Abcam AB193203); and anti-PLK1 (Santa Cruz, SC-17783). USP16 plasmids were described before (27 (link)). USP16-T203AS214A(2A) plasmids were generated using specific primers (sequences available upon request) following the manufacturer’s instructions (ClonExpress Ultra One Step Cloning Kit, Vazyme C115). OGT plasmids and antibodies have been described (31 (link)).

Antibodies used in this study included: anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and anti-GST (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-HA (Bethyl, Montgomery, TX, USA), anti-CCT4 (Abcam, Cambridge, United Kingdom), and anti-FLAG (Sigma, St. Louis, MO). All fluorescence-conjugated secondary antibodies were from Invitrogen.