Fig. 8. Expression of GAR22 proteins in mammalian tissues and cells. (A) Western blotting of mouse tissues. Proteins extracts from various mouse tissues were resolved on an SDS-PAGE gel, transferred onto Immobilon membrane, and probed with polyclonal anti-GAR22 C15 antibody. mGAR22ß was detected in testis and, at a lower level, in brain (indicated with arrowhead). No mGAR22 protein could be detected in any of the tissues. The sizes of molecular mass markers are in kDa. (B) GAR22 immunoprecipitation from growing or arrested cultured cells. GAR22 protein was precipitated with C15 antibody from either proliferating (P) or growth-arrested (A) cells, run on an SDS-PAGE gel, transferred onto Immobilon membrane and probed with C15 antibody. Expression of GAR22ß was induced by growth arrest in all four cell lines. No GAR22a could be precipitated from any of the cell lines. CAD cells were arrested by serum starvation for 48 hours. COS7, NIH 3T3 and GC4 cells were arrested by contact inhibition for 48 hours. (C) Time course of GAR22ß induction in growth-arrested CAD cells. GAR22 protein was immunoprecipitated with the C15 antibody from CAD cells serum starved for indicated periods of time. The precipitated protein was visualized by western blotting with C15 antibody. (D) Phosphatase treatment of endogenous GAR22ß. GAR22 protein was immunoprecipitated with C15 antibody from serum-starved (48 hours) CAD or COS7 cells and treated with either the serine/threonine phosphatase PP1 or the tyrosine phosphatase TCP. The reactions were run on an SDS-PAGE gel, transferred onto Immobilon membrane and probed with C15 antibody. A mobility shift was observed only in the samples treated with PP1.

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