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. 2020 May 4;130(6):2978–2991. doi: 10.1172/JCI132366

Figure 2. Tat–Beclin 1–treated CMs show decreased levels of cellular membranes.

Figure 2

(A–D) NRCMs were treated with Scrambled or Tat–Beclin 1 (5 and 10 μM) for 3 hours and subjected to membrane fractionation assays. The heavy membrane and cytosolic fractions were analyzed by Western blotting using anti-calnexin (ER), anti-PDH (mitochondria), anti-PMCA (plasma membrane), and anti–α-tubulin antibodies (A). Expression ratios of calnexin (B), PDH (C), and PMCA to α-tubulin (D) were quantified; mean ± SD, n = 5 (B), n = 4 (C and D); *P < 0.05, **P < 0.01, 1-way ANOVA with Dunnett’s post hoc test. (E and F) NRCMs were transfected with siControl (siCtrl) or siVAPA and siVAPB. After 60 hours, cells were treated with Scrambled or Tat–Beclin 1 at the indicated doses for 3 hours and subjected to membrane fractionation assays. (E) Whole cell lysates (WCL) and heavy membrane (mem.) fractions were used for immunoblot analyses with anti-calnexin, anti-VAPA, anti-LC3, and anti–α-tubulin antibodies. (F) Cell death induced by 10 μM Tat–Beclin 1 was quantified with CellTiter-Blue assays; mean ± SD, n = 4 values were measured from more than 4 different wells per experiment; **P < 0.01, 1-way ANOVA with Tukey’s post hoc test. See also Supplemental Figure 2.