Skip to main content
. 1999 Aug;19(8):5535–5547. doi: 10.1128/mcb.19.8.5535

FIG. 1.

FIG. 1

FIG. 1

FIG. 1

FIG. 1

Him1 and Hsk1 proteins form an active kinase complex. (A) Extracts were prepared from insect cells expressing Hsk1 (WT, wild-type; KK, KK129, 130RS mutant) with or without HA-tagged Him1 protein as indicated in the figure, and immunoprecipitates made with anti-Hsk1 antibody or anti-HA antibody were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis through Western blotting with the indicated antibody. Immunoprecipitate from the extract expressing both the wild-type Hsk1 and HA-tagged Him1 with anti-Hsk1 antibody was incubated with (lane 10) or without (lane 9) the mixture of λ phosphatase and calf intestine alkaline phosphatase for 60 min at 30°C before being applied to SDS-PAGs. (B) Concentrated extracts were prepared from the wild-type fission yeast cells as previously described (46) and then immunoprecipitated with anti-Him1 antibody (lane 2), anti-Hsk1 antibody (lane 3), or a control antibody (lane 4), followed by Western blotting with the anti-Him1 antibody. Lane 1, an insect cell extract expressing HA-tagged Him1 protein lacking the N-terminal 13 amino acids. The immunoprecipitates (ppt) (lanes 5, 7, 9, and 11) or their supernatants (sup) (lanes 6, 8, 10, and 12) prepared from the same extract were analyzed by Western blotting with anti-Hsk1 (lanes 5 to 8) or with anti-Him1 (lanes 9 to 12) antibody. (C) The anti-HSK immunoprecipitates used in panel A were assayed in kinase reactions in the presence of GST-SpMCM2N protein as a substrate as described in Materials and Methods (lanes 1 to 8). Immunoprecipitates were prepared with anti-HA antibody from extracts expressing HA-tagged Him1 with or without Hsk1 and were used for kinase assays in the presence of GST-SpMCM2N protein (lanes 9 to 11, upper panel). The presence of Him1 or Hsk1 protein was examined by Western blotting (lanes 9 to 11, middle and lower panels). (D) Hsk1 (wild-type or KK) and HA-tagged Him1 protein were separately expressed and immunoprecipitated with anti-Hsk1 and anti-HA antibody, respectively. IPs were mixed in vitro as indicated in the figure (lanes 1 to 5). − and +, the absence and the presence of the IP indicated to the left in the reaction mixtures. HA-Him1p represent hyperphosphorylated HA-tagged Him1 protein. Lane 6, IP from extract coexpressing Hsk1 and HA-Him1 proteins. All the reaction mixtures contained GST-SpMCM2N protein as a substrate.