Na _V 1.5 and EB1 could be coimmunoprecipitated reciprocally when expressed in HEK293 cells but not in ventricular myocytes, while FRAP experiments suggested Na _V 1.5/EB1 interactions at the ICD region of myocytes. (A) GFP-Na_V1.5 expressed in HEK293 cells was coimmunoprecipitated with native EB1 reciprocally. WCL was prepared from HEK293 cells incubated with 8CPT-cAMP/okadaic acid for 4 h or time control with 1% Triton lysis buffer. (−) IP lanes were loaded with eluates from protein A/G beads incubated with WCL without antibody. (+) IP lanes were loaded with eluates from protein A/G beads incubated with WCL and immunoprecipitating (IP) antibody: GFP rabbit Ab (left) or EB1 rat Ab (right). The immunoblot (IB) Abs are listed on the left. Double and single blue circles denote EB1 dimer and monomer bands. (B) GFP-Na_V1.5 and EB1-mRFP coexpressed in cardiac myocytes did not coimmunoprecipitate. Experiments validating GFP-Na_V1.5 and EB1-mRFP as surrogates of native Na_V1.5 and EB1 in myocytes are presented in Fig. S4. Shown are protein(s) expressed in myocytes and conditions (CON or PKA; top), proteins loaded in lanes: WCL (prepared with 1% Triton lysis buffer), IP with mCherry “mChr” or GFP rabbit “rab” Ab, and supernatant (WCL after immunoprecipitation; middle), and immunoblot images probed with GFP goat Ab (upper row) or EB1 rat Ab (lower row). Left: Specificity of immunoprecipitation. EB1-mRFP expressed alone could be immunoprecipitated with mCherry Ab but not by GFP Ab, and GFP-Na_V1.5 expressed alone could be immunoprecipitated with GFP Ab but not by mCherry Ab. Right: In WCLs prepared from myocytes coexpressing GFP-Na_V1.5 and EB1-mRFP cultured under CON or PKA conditions for 15 h, mCherry rabbit Ab immunoprecipitated EB1-mRFP but not GFP-Na_V1.5, and GFP rabbit Ab immunoprecipitated GFP-Na_V1.5 but not EB1-mRFP or native EB1. Red star and blue circle denote the band positions of EB1-mRFP and native EB1, respectively. (C) GFP-Na_V1.5 in HEK293 cells was present in Triton-soluble fraction (detected in 1% Triton WCL), while Na_V1.5 in ventricular myocytes was not present in Triton-soluble fraction (undetectable in 1% Triton lane) but could be extracted with 2% SDS RIPA buffer (detected in 2% SDS lane). (D) Contrasting the subcellular environment of Na_V1.5 and EB1 in ventricular myocytes. Shown are immunoblot images of cytosolic and SDS extracted fractions of CON and PKA myocytes (incubation 15 h) probed for Na_V1.5 and EB1. CB stain shows loading levels. The CB stain of the cytosolic fraction is modified from the one shown in Fig. 4 A. (E) Using FRAP to monitor mobilities of GFP-Na_V1.5 and EB1-mRFP expressed in ventricular myocytes. Top left: Representative images of a live myocyte with four ROIs marked: red—cell center, green—cell end, blue—reference in cell area not photobleached, yellow—background in cell-free area. The corresponding time courses of FRAP are plotted below. Background bleach was corrected based on fluorescence decline in ROI 3, and the fluorescence intensity was normalized to between 1 (right before photobleaching) and 0 (the first scan after photobleaching). Bottom: Average time courses of FRAP of GFP-Na_V1.5 and EB1-mRFP. Shown are the mean (colored bright and dark green for GFP-Na_V1.5 or bright and dark red for EB1-mRFP) and standard error (gray) values superimposed on double-exponential fit (black curve). Left most panel illustrates the calculation of “% of fluorescence recovered 2 min after photobleaching.” Top right: Bar graphs (mean and SE) and individual data points of percentage of fluorescence recovered 2 min after photobleaching for GFP-Na_V1.5 and EB1-mRFP measured from cell center and cell end. Listed P values are from t tests between specified groups. Source data are available for this figure: SourceData F9.