Fig. 2

Validation of AGO2 as a CRBN-downstream binding protein. a Expression of His-tagged CRBN and MRP1 antibody 42.4 epitope-tagged AGO2 in BHK cells. Total proteins from whole cell lysates were separated on an acrylamide gel and probed with mouse-anti MRP1 antibody 42.4 (42.4), M-anti-CRBN and M-anti-Actin antibodies. b Co-IP of CRBN with 42.4-tagged AGO2. Samples were IPed with 42.4 and probed with either rabbit-anti-CRBN (R-anti-CRBN) or rabbit-anti-AGO2 (R-anti-AGO2). c Co-IP of 42.4-tagged AGO2 with CRBN. The three cell lysates mentioned above were IPed with our mouse-anti-CRBN antibody 2F11G5 and probed with either rabbit-anti-CRBN or rabbit-anti-AGO2. d & e Co-IP of wild-type AGO2 with endogenous CRBN in JJN3 cell (d) and in My5.LV cell (e). Samples were IPed with our mouse-anti-CRBN antibody 2F11G5 and probed with either rabbit-anti-AGO2 or rabbit-anti-CRBN. Input: whole cell lysates; IP: whole cell lysates immunoprecipitated (IPed) with M-anti-CRBN; Control: whole cell lysates IPed with protein G beads without adding primary antibody; IB: samples probed with the antibodies indicated in the figure