Fig. 4. TNGW1 and GW182 are independent products of human TNRC6A gene. (A) Expression of both TNGW1 and GW182 were detected in HeLa cells. GWB components were enriched by IP using human anti-GWB serum 18033 and analyzed by western blotting using rabbit anti-GW182 antibodies 5182 and 6642, mouse anti-GW182 mAb 4B6 and anti-rTNR mAb 2E11. All three anti-GW182 antibodies recognized two bands of 210 and 180 kDa, whereas anti-rTNR 2E11 only recognized the 210-kDa band regarded as TNGW1 – the novel isoform of GW182. (B) TNGW1 was detected by multiple anti-rTNR antibodies. IP samples from serum18033 were also examined by rabbit polyclonal antibodies 6225, 6226, and mouse mAb 5C8 recognizing the N-terminal domain rTNR. A 210-kDa band was detected by each of these antibodies regarded as TNGW1. (C) TNGW1 knockdown using siRNA specifically targeting TNGW1 did not affect the level of GW182 in HeLa cells. HeLa cells were transfected with siRNA specifically targeting the TNGW1 isoform (siTNR) or siRNA specifically targeting both isoforms (siGW182) using lipofectamine 2000 (LP 2000). After 48 hours of transfection, cells were lysed and analyzed by western blotting using rabbit anti-rTNR serum 6225 or anti-GW182 serum 5182. Compared with mock transfected and untreated control groups, siGW182 knocked down both TNGW1 and GW182, whereas siTNR only knocked down TNGW1.