Precipitation with TJP1 and ASS1. The faint band observed observed for EB2 inside a CGN immunoprecipitation lane led us to consider it might be due to weak and for EB2 3-Methyl-2-cyclopenten-1-one References within a CGN immunoprecipitation lane led us to think about it may be as a consequence of weak and indirect indirect association with proteins from any CGN protein complex. We didn’t observe VCL in CGN association with either buffer any CGN protein complex. We did not observe VCL it may immunoprecipitates inproteins from situation (Figure 3 and information not shown) even thoughin CGN nonetheless immunoprecipitates in either buffer condition (Figure three and information not shown) even though it might nonetheless bind CX26 by means of an additional intermediate interactor. While CX43specific blot band migrates bind CX26 via an further intermediate interactor. Although CX43specific blot band migrates quite close to an unspecific band observed within the adverse manage, its coimmunoprecipitation with extremely close to an unspecific band observed within the unfavorable manage, its coimmunoprecipitation with CGN is demonstrated (Figure 3). Lastly, we show that CGN coimmunoprecipitates with CX30 and CGN is demonstrated (Figure three). Lastly, we show that CGN coimmunoprecipitates with CX30 and CX31 (Figure 3). CX31 (Figure 3).Figurewith CX26, CX30, CX31, CX43, EB2, TJP1, and ASS1, which can be indicated by the respective arrows. No three. Immunoprecipitation of cingulin from adult mouse liver and its coimmunoprecipitation with CX26, CX30, CX31, CX43, observed with VCL. The protein molecular mass respective arrows. coimmunoprecipitation is EB2, TJP1, and ASS1, which can be indicated by the is indicated in No coimmunoprecipitation is observed with VCL. The protein molecular mass is indicated in kiloDaltons (kDa). kiloDaltons (kDa).Figure 3. Immunoprecipitation of cingulin from adult mouse liver and its coimmunoprecipitationInt. J. Mol. Sci. 2018, 19, 2535 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW77 of 20 ofThe adult mouse liver was colabeled for Cx26 and three adaptor proteins referred to as CGN, FLNB, The adult mouse liver was colabeled for Cx26 and three adaptor proteins called CGN, or DAAM1. The four proteins have been detected in hepatocytes and in patterns constant with plasma FLNB, or DAAM1. The four proteins were detected in hepatocytes and in patterns constant with membrane localization (Figure 4, arrowheads) as nicely as cytoplasm organelles (Figure four). Couple of plasma membrane localization (Figure 4, arrowheads) also as cytoplasm organelles (Figure four). overlapping signals have been observed in hepatocytes for CX26 and DAAM1 at the plasma membrane Couple of overlapping signals have been observed in hepatocytes for CX26 and DAAM1 at the plasma membrane (Figure four, arrows). The paucity of these observations led us to exclude colocalization of those (Figure four, arrows). The paucity of these observations led us to exclude colocalization of these proteins. proteins. CX26 colocalization with CGN or FLNB was not observed under those conditions. CX26 colocalization with CGN or FLNB was not observed below those situations. Furthermore, we show In addition, we show that all 3 adaptor proteins which includes CGN, FLNB, and DAAM1 are expressed that all 3 adaptor proteins including CGN, FLNB, and DAAM1 are expressed in crucial regions of in crucial regions of P14 mouse cochlea for instance the organ of Corti (OC), stria vascularis, spiral Tunicamycin Fungal ligament, P14 mouse cochlea which include the organ of Corti (OC), stria vascularis, sp.