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. 2015 Jun 2;11(6):810. doi: 10.15252/msb.20145880

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© 2015 The Authors. Published under the terms of the CC BY 4.0 license

This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Validation of BCL10 linear ubiquitylation

A, B Validation of BCR stimulation-dependent linear ubiquitylation of BCL10. BCL10 was immunoprecipitated from BCR-stimulated and unstimulated control A20 cells and immunoblotted with LUB9 antibody that binds to linear ubiquitin (A, the upper panel). From the same cell lysates, linear ubiquitylated proteins were isolated using Met1-SUB and probed with BCL10 (A, the lower panel). The latter approach was also used to confirm BCR-induced linear ubiquitylation of BCL10 in A20.2J cells (B). The blots at the bottom of the figure show the expression levels of BCL10 and actin in the input material used for BCL10 immunoprecipitation and Met1-SUB pull-downs.

C BCL10 ubiquitylation is sensitive to linear and K63 linkage-specific deubiquitylases. Linearly ubiquitylated proteins were isolated from BCR stimulated cells with Met1-SUB, treated with the indicated deubiquitylases, and subsequently immunostained with antibodies recognizing BCL10, or ubiquitin. The blots at the bottom of the figures show the expression levels of BCL10 and actin in the input material used for Met1-SUB pull-downs.