Figure 6

Effect of Herpud1 siRNA on polyubiquitin levels and cytosolic and nuclear Ca²⁺ levels in cultured NRVM. (a) Representative Western blots for lysine 48-linked polyubiquitin (Poly-Ub) in control (UNR) and Herpud1-knockdown NRVM (siHerpud1 #2, 200 nM, 48 h) in the presence or absence of the proteasome inhibitor MG-132 (4 μM, 24 h). Graphs correspond to the quantification of each lane, using β-tubulin as a loading control (n = 6). (b) Graph of cytosolic Ca²⁺ concentration kinetics and quantification of the area under the curve (n = 3). (c) Graph of nuclear Ca²⁺ kinetics and quantification of the area under the curve (n = 3). Both (b) and (c) correspond to control (UNR) and Herpud1-knockdown NRVM (siHerpud1 #2, 48 h). (d) Graph of cytosolic Ca²⁺ concentration kinetics and quantification of the area under the curve, in control (UNR) and Herpud1-knockdown NRVM (siHerpud1 #2, 48 h) in the absence or presence of xestospongin C (XeC, 100 μM, 30 min) (n = 3). The black arrow shows the time of addition of histamine (100 mM). Mean ± SEM analyzed using a t-test or one-way ANOVA followed by Bonferroni’s post-test. *p < 0.05 vs. UNR, ^† p < 0.05 vs. UNR + MG-132; ^# p < 0.05 vs. same condition without MG-132, ^‡p < 0.05 vs siHerpud1.