Xenbase Image ID: 155914

Fig. 2. Mid1 physically interacts with Pax6. (A) HeLa cells were transfected with pax6-flag alone or combined with myc-mid1 and analyzed by immunohistochemistry. Nuclei were stained with DAPI. Pictures are shown in false colors: green for Pax6, red for Mid1, blue for nuclei (Bottom). (B) HEK293 cells were transfected as in A. Cytosolic proteins were extracted with hypotonic buffer. Soluble nuclear proteins were extracted from the remaining fraction with high salt buffer. Blot shows Mid1 and Pax6 in the cytosolic (Cyt.) and nuclear (Nuc.) fractions, as well as the reprobes with anti–β-tubulin or anti-topoisomerase 1 antibody to control purity. (C) Interaction of Mid1 with Pax6 was assessed by coimmunoprecipitation performed in HEK293. (Left) Eluted myc-tagged proteins after immunoprecipitation (upper blot) and precipitated Pax6 (lower blot, reprobed with anti-Pax6 antibody). (Right) Blots for the input proteins. (D) Interaction of Mid1 and Pax6 was verified by GST using purified GST-Pax6 or purified GST as a control and lysates of HEK293 cells transfected with myc-mid1. (Left) Eluted myc-tagged proteins after GST pull down (upper blot) and precipitated Pax6 (lower blot, reprobed with anti-Pax6 antibody). (Right) Input protein of the lysate. (E) Pax6-flag was expressed alone or together with myc-Mid1 in HEK293 cells in the presence or absence of the proteasome inhibitor lactacystin (Lac), the E1 activating enzyme inhibitor Pyr41, or the caspase inhibitor Z-VAD-FMK (Z-VAD) as indicated. For loading control, blots were reprobed with an anti-GAPDH antibody. 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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