Ube3a

Western blots were carried out using the primary antibodies; P-ERK (Cell Signaling), ERK (Cell Signaling), Tubulin (Sigma), UBE3A (Bethyl Laboratories), ELK1 (Epitomics) and p53 (Calbiochem). Proteins were detected using infrared dye-conjugated secondary antibodies (LI-COR Bioscience, IRDye 800CW and IRDye 680LT). The signal was collected with a LI-COR Odyssey Infrared Imager. Data were quantified using Odyssey software (LI-COR Bioscience, Odyssey Infrared Imaging system application software version 3.0.25). For microscopy, cells were grown on coverslips and fixed in 3.7% Paraformaldehyde for 15 minutes. DNA was staining with Hoechst. Images were acquired on a Delta Vision RT (Applied Precision) restoration microscope using a 100x/1.40 Plan Apo objective. The images were collected using a Coolsnap HQ (Photometrics) camera with a Z optical spacing of 0.2 μm. Raw images were then deconvolved using the Softworx software and maximum intensity projections of these deconvolved images are shown in the results.

Immunoblotting was performed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as previously described⁶² . Antibodies used in immunoblotting include: Chk1 phospho-S317, Mcm2, Ube3a, Incenp, Smc2 antibodies from Bethyl Laboratories (Montgomery, TX); B55α antibody from Abcam (Cambridge, MA); β-actin, phospho-Cdk substrate and Plk1 antibodies from Cell Signaling Technology (Beverly, MA); Mastl antibody from Millipore (Billerica, MA), and phospho-RPA S4/8 and S33 antibodies as previously characterized^{57 (link)}. Xenopus Cdc25 antibody was a gift from Drs. Kumagai and Dunphy (Caltech). For immunoprecipitation, anti-mouse or anti-rabbit magnetic beads (New England Biolabs) were conjugated to primary antibodies, and then incubated in cell lysates for 1 h. The beads were collected using a magnet, washed, eluted with Laemmli sample buffer, and analyzed by immunoblotting.

The following primary antibodies were used: Tac7G7 (7G7B6, ATCC, HB-8784), SK2 (Alomone, APC-028), SK2 (Alomone, AGP-045), Ube3a (Sigma, E8655), Ube3a (Bethyl Laboratories, A300–351A), PSD95 (Invitrogen, MA1–045), Rab11 (abcam, ab95375), Ubiquitin (Ub, abcam, ab7780), Ub (Santa Cruz, sc-9133), Phosphoserine (Millipore, ab1603), HA (Sigma, H6908), EEA1 (abcam, ab2900), LAMTOR4 (Cell signaling technology, 12284), and β-actin (Sigma, A5441). All secondary antibodies for Western blots were obtained from LI-COR, and for immunofluorescence Alexa-488, −594, and −633 conjugated secondary antibodies were obtained from Invitrogen. Forskolin was obtained from Sigma; apamin was obtained from Millipore; Ro 20–1724 was purchased from Santa Cruz, and KT5720, CNQX, and D-AP5 were purchased from Tocris.