Xenbase Image ID: 155915

Fig. S2. Mid1 reduces endogenous Pax6 protein. (A) Mid1 physically interacted with Pax6. HEK293 cells were transfected with GFP-pax6 + myc-mid1 or myc-mid1 alone. The left panels show eluted Pax6 protein after immunoprecipitation with an anti-Myc antibody (upper blot) and precipitated myc-Mid1 (lower blot, reprobe with anti-Myc antibody). The right panels show blots with the respective lysates for the input proteins. (B) Xenopus animal cap explants expressed Pax6 upon neuralization by microinjection of 400 μg synthetic RNA of the BMP4 inhibitor noggin. Approximately 30 animal cap explants were separated in each lane. Coinjection of Xenopus mid1 RNA (1 ng) led to a reduction of Pax6 protein abundance. The reduction of Pax6 protein levels was considerably stronger, when Mid1 protein was forced to enter the nucleus by the fusion of a nuclear localization signal (mid1nls). The mouse lens epithelial cell line αTN4-1 expresses pax6 endogenously. Transfection of mid1 and of mid1nls led to a reduction of endogenous Pax6 protein. Corresponding Western blots were quantified with the help of ImageJ and normalized against GAPDH. (C) Pax6 levels increased upon proteasome inhibition. αTN4-1 cells were grown in DMEM medium (10% FCS) and were treated with DMSO (−) or with MG132 (5 μM) (+). Pax6 levels increased upon MG132 treatment (+), as well as polyubiquitinated proteins. Anti-GAPDH was used as a loading control. Densitometry was performed using ImageJ (right graph). Image published in: Pfirrmann T et al. (2016) Copyright © 2016. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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