Fig 2: Mitochondria-associated membrane (MAM) proteins are mis-localized in the absence of BOK by immunofluorescence(A–C) Localization in a single plane by confocal microscopy of MitoTracker (first column) with respect to (A) FACL-4, (B) IP3R1, and (C) IP3R2 (second column). The merged image is shown at low (third column) and high (fourth column) power. (D) Quantification of percent co-localization of the individual protein with MitoTracker in 20–30 images per condition from three independent experiments. Data are represented as mean ± SD. Significance was calculated using an ANOVA test (*p < 0.05).See also Figure S3.

Fig 3: Regulation of MAM enriched proteins in obesity(A) Schematic illustration of functional and structural MAM proteins. (B) Western blot and image J based quantification analysis of indicated proteins in liver total lysates from wt/ob/ob and lean/HFD-fed (16 weeks) mice. Arrow indicates the specific Sig1R band, n=3 representative of 2–3 independent experiments. (C) Western blot analysis of indicated proteins of subcellular fractions from mouse livers. CM: crude mitochondria. PM: Pure mitochondria, ER: endoplasmic reticulum and MAM: mitochondria associated ER membranes. Cytochrome C (Cyto C) was used as a mitochondrial marker; IP3R and SERCA were found mostly in the bulk ER. PDI and Calnexin were equally distributed between ER and MAM, and Sigma 1 receptor (Sig1R) enrichment served as a known MAM marker. This figure is representative of at least 5 different preparations (D) Western blot and quantification analysis of indicated proteins in MAMs from wt/ob/ob and lean/HFD mice, n=3. PDI served as a loading control. All the graphs represent mean ± SEM, *P < 0.05, Student’s t-test.

Fig 4: Disruption of the BOK-IP3R interaction decreases MAM proteins with minimal impact on ER-mitochondrial apposition(A) Representative western blot analysis (n = at least 3) for MAM proteins in WT and Bok-/- MEFs as well as Bok-/- MEFs with stable expression of WT FLAG-BOK (+WT) or L34G FLAG-BOK (+L34G). Equal total protein was loaded for each fraction as described in Figure 4. Molecular weight markers are shown in kDa on the left.(B) Representative transmission EM images of Bok-/- MEFs stably transfected with WT (left) or L34G (right) FLAG-BOK. The bottom row shows bounding image of the middle row with mitochondria in red and ER in green as completed in ImageJ.(C) The top scatterplot shows the distance between the mitochondria and ER in WT (n = 125 sites), Bok-/- (n = 117 sites), Bok-/- + WT FLAG-BOK (n = 127 sites), and Bok-/- + L34G FLAG-BOK (n = 124 sites) MEFs from more than 30 EM images. The bottom scatterplot shows the length of each contact site for those that are less than 15 nm between the ER and mitochondria in WT (n = 27), Bok-/- (n = 25), Bok-/- + WT FLAG-BOK (n = 38), and Bok-/- + L34G FLAG-BOK (n = 32) MEFs. WT and Bok-/- MEF data are from Figure 2C. Data are represented as individual values and mean (bar). Significance was calculated using an ANOVA test (ns, not significant).See also Figure S6.

Fig 5: Effect of Gly on the IP3R1 and VDAC1 cellular distributions in porcine oocytes treated with TG. (A, C) IP3R1 cellular distribution pattern after TG treatment or Gly supplementation. IP3R1 (green), DNA (blue), and merged images in MII oocytes. (B, D) VDAC1 cellular distribution pattern after TG treatment or Gly supplementation. VDAC1 (green), DNA (blue), and merged images in MII oocytes. Number of oocytes per group, IP3R1 (Control = 42; TG = 43; TG + 6 mM Gly = 49), VDAC1 (Control = 49; TG = 46; TG + 6 mM Gly = 40). (E) Total RNA was extracted from COCs, and the expression of the IP3R1, VDAC1 and Grp75 was determined by real-time PCR (a, b p < 0.05).