(A) Yeast two hybrid (Y2H) assay with Gal4 activation domain (AD) fusions (prey) and Gal4-DNA binding domain (DB) fusions (bait) indicated in the first two columns. Empty indicates vector only. Growth of two independent co-transformants is shown, and the relative strength of interaction is indicated by -, no interaction, +, weak interaction, ++, strong interaction, and +++, very strong interaction. (B) Co-immunoprecipitation (Co-IP) of in vitro translated (IVT) myc-tagged CYCD2 (upper panel) or CYCD3 (lower panel) incubated with HA-tagged CDKG1, anti-HA beads only (mock), or HA-CDKG1kd (kinase dead mutation). Western blots were done using anti-myc antibodies with input samples (lanes 1,8), IP pellets (P, lanes 2, 4, 6, 9, 11, 13) and IP supernatants (S, lanes 3, 5, 7, 10, 12, 14). (C) Anti-HA Western blot of in vitro translated (IVT) proteins bound to GST or GST-MAT3. HA-CDKG1 (HA-G1, lanes 1, 2), HA-CDKG1 kinase domain only (HA-G1∆N, lanes 3, 4), HA-CYCD3 (HA-D3, lanes 5, 6), HA-CYCD2 (HA-D2, lanes 7, 8) and HA-G1∆N+HA-CYCD2 (lane 9). Schematic structures of IVT proteins are shown on the left with the HA tag indicated by an ellipse. (D) In vitro kinase assay using IVT full length HA-CDKG1 (lanes 2, 4, 6, 8) or a predicted kinase dead version (HA-CDKG1kd, lanes 1, 3, 5, 7) co-immunoprecipitated with (+CYCD3) or without (–CYCD3) IVT CYCD3. Left panel, autoradiograph of proteins separated by SDS-PAGE after the kinase assay (lanes 1–4); right panel, Coomassie blue stained bands corresponding to the autoradiograph (lanes 5–8). Substrate proteins used are labeled on the left. (E) Western blots of anti-HA IP pellets from Chlamydomonas strain mat3-4 complemented with a HA-MAT3 construct (Olson et al., 2010) and synchronized in a 14 hr light/10 hr dark cycle. Anti-HA (top panel) and anti-CDKG1 (bottom panel) blots prepared with extracts from daughter cells (0 hr light), pre-Commitment cells (4 hr light), post-Commitment cells (10 hr light) and S/M phase cells (1 hr dark). (F) Kinase assay using immunoprecipitated HA-CDKG1 or HA-CDKG1kd from Chlamydomonas strain cdkg1-2::HA-gCDKG1 or HA-CDKG1kd. Upper panel, Western blots of total input and IP products. Lower panel, autoradiograph of proteins separated with SDS-PAGE (lane 1 and 2) and coomassie blue stained bands corresponding to the autoradiograph (lane 3 and 4).