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Fig. 1. Generation of the 
N-Zfp36l2 mutant mouse. (A) The strategy used to
disrupt Zfp36l2 is shown with a schematic representation of the
endogenous (12 kb) and targeted (14 kb) alleles of Zfp36l2.
Restriction sites within the wild-type (WT) and targeted gene (S, Sst
I; X, Xba I; E, Eco RV; and N, Not I) are
indicated. The various shadings represent, respectively, 5'-flanking
region of the gene (white), protein coding sequences (black), 5'-UTR and
3'-UTR of the transcript (gray), intron (diagonal-hatch), and PGK-NEO
and PGK-DTA cassettes (cross-hatch). A small double arrow indicates the exon 2
probe used for Southern blot analysis. Black bars indicate the location of the
5' and 3' probes used in Southern blot analysis to select
heterozygous embryonic stem (ES) cells. Genotyping of DNA derived from the
progeny of N-Zfp36l2 heterozygous crosses was performed by PCR and
Southern blot analysis. (B) An example of the PCR results, in which the
0.6 kb and 2.4 kb fragments correspond to WT and Zfp36l2
mutant alleles, respectively. (C) Total DNA (10 µg) was digested with
Sst I and probed with an exon 2 probe; the 10 kb and 2.5 kb
bands correspond to the WT and Zfp36l2 mutant alleles, respectively.
(D,E) Total RNA was isolated from the indicated tissues and bone
marrow-derived macrophages (BMMac) of WT and mutant (Mut) mice and subjected
to northern blot analysis. Each lane in D contains 10 µg of total RNA
probed with Zfp36l2 exon 1 and Gapd probes, as indicated by
the arrows. The positions of the 28S and 18S ribosomal RNAs are indicated. (E)
RNA samples from the same tissues as described in D were subjected to northern
blot analysis and hybridized with Zfp36l2 exon 2 (upper arrow) and
Gapd (lower arrow) probes, showing the presence of a remaining
Zfp36l2 transcript in the mutant mice. (F) Protein extracts from HEK
293 cells overexpressing Zfp36l2 (lane 1), and bone marrow-derived macrophages
from WT and N-Zfp36l2 mutant mice (lane 2 and 3, respectively), were
separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane.
Blotting with the Zfp36l2 amino-terminal peptide antibody revealed a broad
band of 60 kDa corresponding to the predicted size of Zfp36l2 in the
protein extracts from HEK 293 cells overexpressing Zfp36l2 and the cells from
the WT mice, but not from the mutant cells (left panel). In the presence of
competing peptide, the Zfp36l2 signal was blocked (right panel). (G) Western
blot analysis using protein extracts from spleen (left) and BMMac (right) of
WT and N-Zfp36l2 mutant (Mut) mice (lane 2 and 3, respectively) and HEK
293 cells overexpressing Zfp36l2-HA (left panel, lane 1) or not expressing
Zfp36l2 (right panel, lane 4) were probed with a carboxyl-terminal peptide
antiserum. This revealed that the band corresponding to Zfp36l2 exhibited a
smaller size in tissues and cells from the mutant mice (arrow), and that the
levels of expression of the protein were lower in the spleen, but
approximately equal in BMMac. The dotted arrows point to the major
non-specific (NS) bands recognized by this antiserum.
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