Anti myc agarose beads

Detailed information is available in Supplemental Materials and Methods.
Normal human lung fibroblasts (NHLFs) were purchased from Lonza (Walkersville, MD, USA). BEAS-2B cells and HEK293 cells were purchased from Korea Cell Line Bank (Seoul, Korea). Human TGF-β₁ was obtained from R&D Systems (Minneapolis, MN, USA) and other chemicals were purchased from Sigma (St. Louis, MO, USA). Antibodies were purchased from the following companies: anti-E-cadherin, anti-zona occuludens 1 (ZO-1), anti-N-cadherin, anti-vimentin, anti-SMAD2, anti-SMAD3, anti-phospho-SMAD2 (Ser465/467), anti-phospho-SMAD3 (Ser423/425), anti-SMURF2, horse radish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-mouse IgG (Cell Signaling, Beverly, MA, USA), anti-TTC3, anti-hemagglutinin (HA), anti-DYK (FLAG), and anti-Myc antibodies (Sigma), anti-α-SMA antibody (Abcam, Cambridge, MA, USA), anti-ubiquitin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-GAPDH antibody (AbFrontier, Seoul, Korea). Anti-DYK (M2) and anti-Myc agarose beads were purchased from Sigma and Thermo Scientific (Rockford, IL, USA), respectively. Detailed information about antibodies was given in Supplemental Table 1. Reagents including E1 (UBE1), E2 (UBE2E3), and the reaction buffer for the in vitro ubiquitylation assay were purchased from Boston Biochem (Cambridge, MA, USA).

Cells were cross-linked with 1% formaldehyde at OD₆₀₀ ≈ 0.6 to 0.9 for 20 min at room temperature and then quenched with glycine to a final concentration of 300 mM for 5 min at room temperature. Cells were washed twice with cold Tris-buffered saline and lysed with 0.5 mm zirconia beads in FA-lysis buffer (Aparicio et al. 2005 ) with protease inhibitors (Roche) in a MP Fastprep-24. Chromatin was isolated as described (Aparicio et al. 2005 ). For 13XMyc-Sir3 immunoprecipitations, 50 μl of anti-Myc agarose beads (Sigma E6654) were incubated overnight at 4° with sonicated chromatin from 42.5 ml of culture. For Gal4 immunoprecipitations, 4 μl of anti-Gal4 antibody (Abcam 1396) and 25 μl protein A sepharose (GE Healthcare) were incubated with sonicated chromatin under identical conditions. Resin washes, elution, and DNA purification were performed as described (Aparicio et al. 2005 ). Precipitated DNA fragments were analyzed by qPCR, as described previously. The negative-control primer set for 13XMyc-Sir3 ChIP, ARS504, was chosen because it gave a consistently low IP/Input signal indistinguishable from a no-tag control and has been shown not to be bound by Sir3. Gal4 ChIP was normalized to a region downstream of the GAL1 gene not bound by Gal4. ChIP values are presented as enrichment relative to a control locus [(IP(primer)/IN(primer)]/[IP(control)/IN(control)].

Cells were harvested using pre-chilled PBS and lysed using 1% NP-40 lysis buffer with protease inhibitor (Roche). HA-tagged or Myc-tagged fusion proteins were immunoprecipitated by incubation overnight at 4°C with anti-HA or anti-Myc agarose beads (Sigma) and bound protein identified by Western blotting using antibodies against HA (Covance), Myc (BD Biosciences), or CUL1 (Zymed). Antibodies against tubulin (Sigma) or GAPDH (Abnova) were used on the loading controls.