Figure 4. Protein interactions between... | Rockefeller University Press

Figure 4.

$Protein interactions between GluA1, KIF13A and centaurin-α1 upon LTP induction. (A) GFP-immunoprecipitation from protein extracts of 7‒10 DIV organotypic hippocampal slices expressing GFP-KIF13A-ST protein or GFP as control. Extracts were obtained from slices maintained at basal conditions (aCSF) or from slices undergoing cLTP induction for different times (7.5 min, 15 min) or from slices recovered for 15 min following cLTP (Recovery), as indicated. Coimmunoprecipitated proteins (“Bound”) and input fractions were analyzed by Western blot using antibodies for GluA1 and centaurin-α1. IP, immunoprecipitation. Left, representative Western blots. Right, quantification of the amount of coprecipitated GluA1 normalized to baseline (dashed line). Dark symbols represent mean ± SEM; gray symbols represent individual values from each experiment. n = 5, 4, 5, and 4 experiments for baseline, 7.5-min cLTP, 15-min cLTP, and recovery, respectively. (B) Left: Similar to A, with the immunoprecipitation of endogenous GluA1. Nonimmune rabbit IgGs were used as immunoprecipitation control. GluA1, centaurin-α1, and KIF13A were detected by Western blot. Right: Quantification of the coprecipitated amount of KIF13A (green circles) and centaurin-α1 (purple circles). P values (P = 0.009 and P = 0.003) represent significant difference from baseline (Friedman test). n = 4, 4, 4, and 3 experiments for baseline, 7.5-min cLTP, 15-min cLTP, and recovery, respectively, in the case of KIF13A coimmunoprecipitation. n = 5, 5, 5, and 4 experiments for baseline, 7.5-min cLTP, 15-min cLTP, and recovery, respectively, in the case of centaurin-α1 coimmunoprecipitation. (C) Left: Similar to B, with the immunoprecipitation of endogenous centaurin-α1 and Western blot detection of endogenous KIF13A, GluA1, and centaurin-α1. Nonimmune goat IgGs were used as immunoprecipitation control. Right: Quantifications of the coprecipitated KIF13A (green circles) and GluA1 (black circles). P value represents significant difference from baseline (P = 0.009, Friedman test). n = 4, 4, 4, and 3 experiments for baseline, 7.5-min cLTP, 15-min cLTP, and recovery, respectively, in the case of KIF13A coimmunoprecipitation. n = 5, 5, 5, and 4 experiments for baseline, 7.5-min cLTP, 15-min cLTP, and recovery, respectively, in the case of GluA1 coimmunoprecipitation.$

Protein interactions between GluA1, KIF13A and centaurin-α1 upon LTP induction. (A) GFP-immunoprecipitation from protein extracts of 7‒10 DIV organotypic hippocampal slices expressing GFP-KIF13A-ST protein or GFP as control. Extracts were obtained from slices maintained at basal conditions (aCSF) or from slices undergoing cLTP induction for different times (7.5 min, 15 min) or from slices recovered for 15 min following cLTP (Recovery), as indicated. Coimmunoprecipitated proteins (“Bound”) and input fractions were analyzed by Western blot using antibodies for GluA1 and centaurin-α1. IP, immunoprecipitation. Left, representative Western blots. Right, quantification of the amount of coprecipitated GluA1 normalized to baseline (dashed line). Dark symbols represent mean ± SEM; gray symbols represent individual values from each experiment. n = 5, 4, 5, and 4 experiments for baseline, 7.5-min cLTP, 15-min cLTP, and recovery, respectively. (B) Left: Similar to A, with the immunoprecipitation of endogenous GluA1. Nonimmune rabbit IgGs were used as immunoprecipitation control. GluA1, centaurin-α1, and KIF13A were detected by Western blot. Right: Quantification of the coprecipitated amount of KIF13A (green circles) and centaurin-α1 (purple circles). P values (P = 0.009 and P = 0.003) represent significant difference from baseline (Friedman test). n = 4, 4, 4, and 3 experiments for baseline, 7.5-min cLTP, 15-min cLTP, and recovery, respectively, in the case of KIF13A coimmunoprecipitation. n = 5, 5, 5, and 4 experiments for baseline, 7.5-min cLTP, 15-min cLTP, and recovery, respectively, in the case of centaurin-α1 coimmunoprecipitation. (C) Left: Similar to B, with the immunoprecipitation of endogenous centaurin-α1 and Western blot detection of endogenous KIF13A, GluA1, and centaurin-α1. Nonimmune goat IgGs were used as immunoprecipitation control. Right: Quantifications of the coprecipitated KIF13A (green circles) and GluA1 (black circles). P value represents significant difference from baseline (P = 0.009, Friedman test). n = 4, 4, 4, and 3 experiments for baseline, 7.5-min cLTP, 15-min cLTP, and recovery, respectively, in the case of KIF13A coimmunoprecipitation. n = 5, 5, 5, and 4 experiments for baseline, 7.5-min cLTP, 15-min cLTP, and recovery, respectively, in the case of GluA1 coimmunoprecipitation.

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