Fig. 2. Expression of AtMSI1. (A) Seedlings were grown for 7 days on plates containing MS medium, otherwise plants were grown on soil under a long-day light regime. RNA was extracted from different plant organs and RNA blots containing 10 µg of total RNA were probed with an AtMSI1 probe (upper panel). The ethidium bromide-stained agarose gel is shown as a loading control (lower panel). (B) HA-tagged AtMSI1-5 were produced in vitro. Immunoblots containing similar fractions of the total reaction mixture were tested with either affinity-purified a-MSI1 or with a-HA antisera. Extract not supplemented with an AtMSI cDNA served as control (last lane). (C) Protein extracts were prepared from different plant organs. Leaves were harvested just after emergence from the shoot apical meristem and before they were completely expanded (young leaves) or after many other vegetative leaves had developed (mature leaves). Seedlings were grown on plates containing 50% MS medium. 10 µg protein was loaded in each lane. Blots were probed with affinity-purified, a-MSI1-specific antisera. (D) Protein was extracted from leaves of wild-type control plants (Col) and siblings of a segregating progeny of an AtMSI1 overexpression (OE) line (1OEa3). Ten µg protein per sample was subjected to immunoblotting with affinity-purified, a-MSI1-specific antiserum (upper panel). Ponceau red-staining of the blot is shown as a loading control (lower panel). (E) RNA was isolated from leaves of wild-type and AtMSI1-CS plants before bolting. After treatment with DNaseI, RNA was subjected to reverse transcription in the presence or absence of reverse transcriptase using oligo(dT) primers. PCR with different cDNA-specific primers was performed on aliquots of the produced cDNA.