Ha ub

Flag-UCK1 and Myc-KLHL2 were separately transfected into HEK293 cells. 48 h later, Flag-UCK1 and Myc-KLHL2 were purified with antibodies against Flag and Myc, respectively. Then these proteins were added to the reaction mixture containing adenosine triphosphate (ATP), HA-Ub, E1 and E2 (Boston Biochem, MA). The reaction was stopped and IP with Flag antibody and subsequent IB assay were performed to measure co-IP of UCK1.

In vitro ubiquitination assays were conducted as described previously (Yang et al., 2006 (link)) with slight modifications. 1 μg of purified wild type and mutant His-GmPUB1 proteins, 50 ng of human recombinant E1 enzyme (Boston Biochem, Cambridge, MA), 150 ng of E2 UbcH5b (Boston Biochem, Cambridge, MA), 2 μg of HA-Ub (Boston Biochem, Cambridge, MA) were incubated in a final volume of 30 μl reaction buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 0.2 mM DTT, 3 mM ATP, 10 mM creatine phosphate and 0.1 unit of creatine phosphokinase (Sigma-Aldrich, St. Louis, MO). Reactions were incubated at 30°C for 2 h and stopped by boiling with 4X SDS-PAGE loading buffer for 5 min. 15 μl of each reaction was analyzed by electrophoresis on 10% SDS-PAGE and subjected to immunoblot analysis. Ubiquitination was detected with anti-HA antibody (Invitrogen, Carlsbad, CA) and anti-His (Amersham Bioscience, PA) antibody.

SdeC variants were purified as described previously.^{15,47 (link)} Purified GST-Rtn4 was purchased from MRCPPU. Human recombinant monomeric Ub and HA-Ub were purchased from Boston Biochem (R&D systems). All HA derivative peptides were synthesized in Tufts University Core Facility. For further details about protein expression and purification, please see the Electronic Supplementary Information.

To assay for Ptch1 ubiquitination in vivo,
Smurf1^−/−/Smurf2^flox/floxMEFs were infected with either Ad-GFP or Ad-Cre adenovirus for 24 hr, then
transfected with Ptch1-FLAG or Ptch1Δ2PY-FLAG along with HA-Ub using
Lipofectamine Plus (Invitrogen). The cells were lysed 24 hr later and Ptch1 and
its mutant were isolated with anti-FLAG agarose beads and resolved by SDS-PAGE
on 6% or 4–12% gradient gels. The ubiquitinated Ptch1 was then detected
with anti-HA (Roche-Shanghai, China). To assay for Ptch ubiquitination in vitro,
an ubiquitination assay was modified from a previously described procedure
(Tang et al., 2011 (link)). Ptch1-FLAG was
captured from transfected HEK293 cell lysates using anti-FLAG agarose. After a
thorough wash, the Ptch1-bound agarose was divided into three aliquots. Empty
anti-FLAG agarose was used as a control. The in vitro ubiquitination assay was
performed by incubating either Ptch1-bound agarose or control agarose at
37°C for 1 hr with ubiquitin-activating enzyme UBE1, E2-conjugating enzyme
UbcH5c, HA-Ub and ATP (all from Boston Biochem, Cambridge, MA) in the presence
or absence of purified His6-Smurf2 or His6-Smurf2CG. After the incubation, the
supernatant was removed, the agarose thoroughly washed, and the Ptch1-FLAG
eluted using the FLAG peptide (Sigma). The eluted fraction was then subjected to
Western blot analysis.

HeLa cells were transfected with an expression plasmid encoding Myc or Myc-tagged Peli1 or Peli1 deletion mutants, Flag-tagged BubR1 and HA-tagged ubiquitin (HA-Ub) in the indicated combinations. The cells from each plate were collected into two aliquots. One aliquot was used for conventional immunoblotting, and the remaining cells were used for immunoprecipitation of the BubR1 protein complex. Immunoprecipitates were washed three times with TNN buffer, and bound proteins were immunoblotted with the indicated antibodies. Purified GST or GST-BubR1 (200 ng) was incubated with purified His-Peli1 (1 μg) in conjunction with E1 (50 ng UBE1, Boston Biochem), E2 (400 ng UncH13/Uev1a, Boston Biochem), and HA-tagged ubiquitin (2 μg HA-Ub, Boston Biochem) in ubiquitin reaction buffer [5 mM Tris-HCl (pH 7.5), 2 mM MgCl₂, 2 mM ATP and 100 mM NaCl]. Reaction mixtures were incubated for 2 hr at 37 °C and analysed by immunoblotting with anti-BubR1 or anti-ubiquitin and anti-Peli1 antibodies.