Fig. 2.

KLHL14-dependent ubiquitylation and down-regulation of BCR subunits. (A, Top) Western blot analysis of immunoprecipitated endogenous KLHL14 in TMD8 cells. IgG antibody immunoprecipitates = negative control; IP, immunoprecipitated; WCL, whole cell lysates. (A, Bottom) Western blot analysis of immunoprecipitated empty vector (EV), FLAG-HA-tagged KLHL14^WT, two KLHL14 mutant isoforms in TMD8 cells. (B) In vitro ubiquitylation reaction of immunopurified FLAG-tagged KLHL14^WT (Top) and KLHL14^Q255X mutant (Bottom). Ubiquitin reaction buffer contains E1, E2 (UbcH5c), ATP, and ubiquitin. Ub, ubiquitin. (C) Mass-spectrometry measurements of relative protein abundance versus relative ubiquitinated protein abundance upon KLHL14 expression in TMD8 cells. BCR subunits, BCR-associated negative regulators, and Cullin3 are labeled red, blue, and green, respectively. (D, Left) Western blot analysis of whole cell lysates from TMD8, OCI-LY10, and RIVA cells retrovirally transduced with cDNAs encoding EV, FLAG-HA-tagged KLHL14^WT, or KLHL14^Q255X mutant. (D, Right) Quantification of BCR protein levels. (E) FACS analysis of surface IgM and CD79B expression on TMD8 and RIVA cells retrovirally transduced with cDNAs encoding EV, KLHL14^WT, or KLHL14^Q255X mutant coexpressing the LYT2 surface marker. Error bars represent SD of triplicates, and data are representative of three independent experiments.