Figure 8
The T1-LIR 32 kDa and 50 kDa species are enriched by immunoprecipitation (IP) in transduced HEK293T and wild type newborn brain nuclear extract. (a) Total cellular extracts from HEK293T cells transduced with Ad-V5/Thap1-GFP were subjected to immunoprecipitation using anti-V5 antibody, separated by PAGE, and transferred to nitrocellulose. The membrane was incubated with Proteintech anti-Thap1 and anti-V5 antibody. On the right panel, Ponceau S staining of the membrane shows relative protein loading in each lane. Control = untreated cells. (b) Nuclear and cytoplasmic extracts from P1 forebrain were subjected to immunoprecipitation using Proteintech antibody or the SC3H3 antibody. The T1-LIR species of 32 kDa (*) and a very small amount of the 50 kDa species (arrow) can be detected in the elution with the Proteintech (PT) antibody. IP with Santa Cruz 3H3 (SC) (last lane) confirms the enrichment of the 32 kDa species. (c) A protocol similar to that employed in (b) was used except that the P1 forebrain extract was treated with calf intestinal phosphatase (CIP) prior to IP. IP was perfomed using Proteintech (PT) or Santa Cruz (SC) antibodies. Note that the 32 kDa T1-LIR species is obscured by the light chain in the eluate, but the 50 kDa T1-LIR species (arrow) is more easily identified in nuclear samples treated with CIP (compare to 8b).