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. Author manuscript; available in PMC: 2019 May 9.
Published in final edited form as: Kidney Int. 2018 Jan 6;93(4):871–880. doi: 10.1016/j.kint.2017.10.018

Figure 1 |. Specificity of the antibodies against the B1 and B2 subunit isoforms, detection of both subunit isoforms in urinary exosomes, and comparison of the centrifugation versus precipitation methods.

Figure 1 |

(a) Lysates of yeast transformed with empty vector (lane 1) or expression vectors containing the human B1 (lane 2) or B2 subunit (lane 3). Upper and middle panels display signals obtained with B1- and B2-specific antibodies, respectively. Lower panel shows corresponding Ponceau staining. (b) Kidney lysates of wild-type (WT) (lane 1) and B1 knockout (KO) mice (lane 2). Upper and middle panels display signals obtained with B1- and B2-specific antibodies, respectively. Lower panel shows corresponding Ponceau staining. (c) Urinary exosomes isolated from 3 healthy subjects, blotted with B1 (upper panel) or B2 (middle panel) antibody or an antibody for the housekeeping exosomal marker alix (lower panel). (d) After clearing (17,000 g for 15 minutes at 24 °C), the same urine sample was subjected to parallel differential centrifugation and total protein precipitation using a modified method of Wessel-Flügge. The resultant proteins were loaded equally and immunoblotted for B1 and the housekeeping exosomal marker CD9. PTEF, emtpy yeast vector p426TEF. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.