Journal: Communications Biology
Article Title: Doxorubicin induces caspase-mediated proteolysis of KV7.1
Figure Lengend Snippet: Cleavage of K v 7.1 in physiology and pathophysiology. a Representative current traces for K v 7.1-MYC and K v 7.1-D459A-MYC, both coexpressed with KCNE1. b Mean currents amplitude was plotted versus voltage to obtain current−voltage (I−V) relationships in cells expressing K v 7.1-MYC ( n = 39 for vehicle, n = 16 for staurosporine treatment) or K v 7.1-D459A-MYC ( n = 27 for vehicle, n = 17 for staurosporine treatment) and KCNE1 treated with 500 nmol per L staurosporine for 10–12 h. Statistics were tested with two-way ANOVA followed by Bonferroni post-tests. c Immunoblot analysis of HeLa cells coexpressing Kv7.1 with KCNE1-MYC treated with 1 µM staurosporine for 4.5 h. Untransfected (Ø) and vehicle-treated cells served as negative controls. d Biotinylating study analyzed by immunoblots of Hek 293 cells coexpressing K v 7.1 and KCNE1-MYC treated with 1 µM staurosporine for 3 h. Untransfected (Ø) cells as well as cells not treated with biotin served as negative controls. IP Immunoprecipitation. TL total lysate. e Schematic illustration to highlight the position of G460 and A372 and calmodulin binding site in helix A. Western blot analysis of HeLa cell lysates overexpressing indicated constructs. Untransfected (Ø) and K v 7.1-D459A-transfected cells served as negative controls. Densitometric analysis of four independent experiments of CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. f Coimmunoprecipitation study analyzed by immunoblots of HeLa cells overexpressing wild-type K v 7.1 and the A372D mutant with endogenous calmodulin. IP Immunoprecipitation with anti-K v 7.1 antibody, IB Immunoblot, TL total lysate. Untransfected cells (Ø) served as negative control. c Anti-KCNE1 antibody, anti-caspase 3 antibody. c , d Anti-K v 7.1 antibody, anti-GAPDH antibody. e Anti-β-actin antibody. e , f Anti-MYC antibody. f Anti-calmodulin antibody. All graphs are shown as mean and error bars as SEM
Article Snippet: The following antibodies were used: rabbit anti-β-actin (A2066, Sigma-Aldrich, St. Louis, USA), rabbit anti-caspase 3 (8G10, Cell Signaling, Cambridge, UK), rabbit anti-calmodulin (ab45689, Abcam, Cambridge, UK), rabbit anti-Eef2 (eukaryotic translation elongation factor 2, ab33523, Abcam, Cambridge, UK), mouse anti-GAPDH (MAB374, Millipore, Billerica, USA), rabbit anti-KCNE1 (APC-163, Alomone Labs, Jerusalem, Israel), rabbit anti-Kv 7.1 (ab77701, Abcam, Cambridge, UK), mouse anti-MYC (9B11, Cell Signaling, Cambridge, UK), goat anti-MYC (GTX29106, GeneTex Inc., Irvine, USA).
Techniques: Expressing, Western Blot, Immunoprecipitation, Binding Assay, Construct, Transfection, Mutagenesis, Negative Control