Journal: iScience

Article Title: Contact sites between endoplasmic reticulum sheets and mitochondria regulate mitochondrial DNA replication and segregation

doi: 10.1016/j.isci.2023.107180

Figure Lengend Snippet:

Article Snippet: The following antibodies were used: CLIMP63 (Rb, Abcam, ab84712, 1:150), CLIMP63 (Mo, ENZO, ENZ-ABS669, 1: 200), RRBP1 (Rb, Novus Biologicals, NBP1-32813, 1:200), TOM20 (Rb, Abcam, ab186735, 1:250), mtTFAM (Mo, Novusbio, NBP1-71648, 1:150), Calnexin (Mo, Millipore, MABF2067, 1:200), RTN4/NOGOA (Rb, Bio-Rad, AHP1799, 1:200), ATP5a (Mo, Abcam, ab14748, 1:150), SYNJ2BP (Rb, Sigma-Aldrich, HPA000866, 1:200), RRBP1 (Mo, Thermo Fisher Scientific, MA5-18302, 1:200), IP3R1 (Rb, Novus Biologicals, NBP2-22458, 1:200), VDAC(Mo, Sigma-Aldrich, MABN504, 1:200).

Techniques: Transduction, Recombinant, Modification, Saline, Transfection, Electron Microscopy, Imaging, In Situ, SYBR Green Assay, Software

Journal: Nature Communications

Article Title: IP 3 receptor isoforms differently regulate ER-mitochondrial contacts and local calcium transfer

doi: 10.1038/s41467-019-11646-3

Figure Lengend Snippet: Ca 2+ flux through the IP3R is dispensable for supporting the ER-mitochondrial contacts. a IP 3 dependent Ca 2+ signaling was tested as in Fig. . in TKO cells rescued with WT rIP3R1 (R1 A) and with a pore-dead mutant IP3R1 (R1 D2550A). Representative cell traces are shown ( N = 30 and 30 cells, respectively, from three independent experiments). b Relative abundance (Mean ± SEM from four independent experiments) of ER, mitochondria and IP3R1 in WT ( N = 45), R1 A ( N = 27) and R1 D2550A ( N = 47) cells plotted against the Z -axis. c Co-distribution (mean, dashed lines) of ER and mitochondria calculated as in Fig. . d Residual sum of squares for mitochondria and ER (mean, dashed lines). e Cumulative abundance of IP3Rs in the 0–1 µm Z sections of each cells (mean, dashed lines). Black lines indicate p < 0.05 with one-way ANOVA with post-hoc Holm’s Method. f Bar charts show the relative frequencies (Mean ± S.E.M of three experiments) of the occurrence of <100 nm interface gap widths between the ER and mitochondrion ( N = 531, 591, 477, and 561 mitochondria for TKO, WT, R1 A, and R1 D2550A, respectively). Black lines represent p < 0.05 with Chi-squared test. g IP 3 dependent Ca 2+ signaling in TKO cells rescued with WT IP3R2, a pore-dead IP3R2 (R2 GS3) and muscarinic acetylcholine receptor M 3 (M3). Representative traces are shown ( N = 164, 189, and 197 cells, respectively, from three independent experiments). h Relative frequencies (Mean ± S.E.M of three experiments) of the occurrence of interface gap widths providing an interface <100 nm between the ER and mitochondrion ( N = 394 (TKO), 268 (R2), 522 (R2 GS3), and 390 (M3) mitochondria were analyzed from three independent experiments). Black lines represent p < 0.01 with Chi-squared test. Source data are provided for panels b , c , d , e , f , h as a Source Data file

Article Snippet: Rabbit polyclonal antibodies recognizing IP3R1 (CT1) (1:5000) raised against the C-terminal 19 amino acid (aa) residues of rat IP3R1 and IP3R2 (NT2) (1:200) raised against N-terminal 320–338 aa residues in mouse IP3R2 (custom made, Pocono Rabbit Farms and Laboratories), mouse monoclonal antibody against 22–230 aa residues of human IP3R3 (1:1000, BD Transduction laboratories Cat# 610312), Calnexin rabbit polyclonal antibody (1:1000, Enzo Cat# ADI-SPA-860), and mouse monoclonal Flag antibody (1:5000, Sigma Cat# F1804-200UG) were used for immunoblotting.

Techniques: Mutagenesis