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. 2020 Mar 3;117(11):6092–6102. doi: 10.1073/pnas.1921187117

Fig. 2.

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 KLHL14WT, two KLHL14 mutant isoforms in TMD8 cells. (B) In vitro ubiquitylation reaction of immunopurified FLAG-tagged KLHL14WT (Top) and KLHL14Q255X 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 KLHL14WT, or KLHL14Q255X 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, KLHL14WT, or KLHL14Q255X mutant coexpressing the LYT2 surface marker. Error bars represent SD of triplicates, and data are representative of three independent experiments.