Generation and confirmation of SOCS3^ΔSB/ΔSB mice. (A) The SOCS3 locus is shown at the top with the targeting construct and targeted allele below. The structure of the knock-in allele after removal of the neomycin selection cassette (PGKNEOpA) by cre-mediated excision is also shown. Exons are indicated as boxes with the coding region shaded. The 120-bp deletion in exon 2 of nucleotides 573 to 692 of the SOCS3 transcript that encodes the SOCS box is indicated by ΔSB. 5′ and 3′ probes used for Southern analysis are indicated. H indicates HindIII; RV, EcoRV. (B) Correct homologous recombination and deletion of the selection cassette was confirmed by Southern blot analyses of tail DNA digested with HindIII using 5′ and 3′ probes. DNA digested with PvuII was probed with a 130-bp SOCS box probe, confirming the absence of this sequence in SOCS3^ΔSB/ΔSB samples. (C) RT-PCR analysis of cDNA prepared from RNA from livers from WT, SOCS3^ΔSB/+, and SOCS3^ΔSB/ΔSB mice injected with IL-6 (5 μg, intraperitoneally 60 minutes prior to the time that they were killed). Primers spanning the SOCS3 SOCS box were used. In SOCS3^ΔSB/ΔSB samples, only the 200-bp transcript lacking the SOCS box was amplified. HPRT was amplified as a loading control. (D) Immunoblot of liver lysates prepared from mice injected with IL-6 (5 μg, intraperitoneally 60 minutes prior to the time that they were killed). SOCS3 was immunoprecipitated with a monoclonal antibody to SOCS3 and immunoblotted using a polyclonal SOCS3 antibody. In SOCS3^ΔSB/ΔSB lysates, only the smaller (20 kDa) SOCS3 protein was detectable. NS indicates nonspecific band.