Mouse anti v5 tag mab

Cell lysates were prepared and subject to immunoprecipitation or/and immunoblot analysis as previously described (15 (link), 46 (link)). The following pAbs and mAbs were utilized: rabbit anti-TRIM56 S4091 pAb (this study); rabbit anti-pSer⁴⁷¹-TRIM56 and anti-pSer⁴⁷⁵ pAbs (this study); rabbit anti-ISG56 pAb (42 (link)); rabbit anti-TRIF pAb (51 (link)); rabbit anti-IRF-3 pAb (52 (link)); rabbit anti-pSer³⁹⁸-IRF3 (Upstate), rabbit anti-MBP pAb (New England Biolabs), mouse anti-TRIM56 mAbs (this study), mouse anti-HA tag mAb (Invivogen); mouse anti-V5 tag mAb (Invitrogen); mouse anti-Flag M2 mAb (Sigma); mouse anti-actin mAb and rabbit anti-calreticulin pAb (both form Sigma); peroxidase-conjugated secondary goat anti-rabbit and goat anti-mouse pAbs (Southern Biotech).

HEK293 cells were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, 4.5 g/l glucose, 584 mg/l L-glutamine, 100 mg/l sodium pyruvate, and 1 U/ml penicillin. Co-transfections were performed with Lipofectamine LTX with PLUS reagents (Life Technologies, Cat#1533810) using full length SKN-1c or WDR-23a cloned into pDEST27 vector and full length SKR-1 cloned into V5-DEST vector. Two days after transfection, cells were lysed with immunoprecipitation lysis buffer (Life Technologies, Cat # 87787) and pulldown was conducted using glutathione-Sepharose 4B at room temperature for 1 h (GE Healthcare, Cat#17-0756-01 Little Chalfont, United Kingdom). Beads from pulldowns were washed extensively with PBS+0.1% Triton-X and eluted with an equal volume of 2X SDS loading buffer by heating at 90°C for 5 minutes. Western blots were carried out as described above, with mouse anti-GST MAb (1:1,000; Santa Cruz Biotech, Cat #B-14 Dallas, TX), and mouse anti-V5 tag MAb (1:1000; Invitrogen, Cat #R960-25).