Ab14734

ER-mitochondrial interactions were assessed using in situ PLA which targeted the complex between the ER inositol 1,4,5-triphosphate receptor (IP3 R)-1 (ab5804, Abcam, Paris, France) and the mitochondrial voltage-dependent anion channel (VDAC)-1 (ab14734, Abcam, UK) as previously described [13 (link)]. In vitro, the assays were conducted on 4% paraformaldehyde-fixed and 0.1% triton-permeabilized primary rat hepatocytes using a green, fluorescent in situ PLA DUOLINK kit (Merck, Darmstadt, Germany). In situ, the assays were carried on in 4 µm paraffin sections of 4% paraformaldehyde-fixed and paraffin-embedded mouse liver samples using a bright-field in situ PLA DUOLINK kit (Merck, USA). Images were analyzed using a custom written Fiji macro on 10 images/sample in 3 to 5 independent series, and the number of VDAC1-IP3 R1 dots was expressed per nucleus.

To determine the monomer/dimer ratio of HSPB1, we first separated mitochondria from cytosol using the standard mitochondrial isolation protocol as described above. Samples were kept on ice all the time, and equal amounts of protein lysate were supplemented with NuPAGE LDS Sample buffer (NP0007, Life Technologies) either with or without 100 mM DTT (20290, Thermo Fisher Scientific). The non-reducing condition retained the disulphide bond between HSPB1 dimers and was visualized using the standard SDS–PAGE protocol using anti-HSPB1 (1:1,000, SPA-800, Enzo Life Sciences), Tubulin (1:10,000, ab7291, Abcam) and VDAC1 (1:1,000, ab14734, Abcam).

Two methods be used for observing mitochondrial morphology in mice brain. A) injection AAV-BBB-Mito DsRed by the tail vein 100 μl/mice. After 4 weeks, mice were anesthetized with pentobarbital sodium (80 mg/ml) and perfused with ice-cold 0.9% saline, finally embedded in OCT (SAKURA#4583). Samples were frozen by liquid nitrogen & isopentane for 45 s and then cut into 100 μm slices. B) Immunostaining. Mice was anesthetized with pentobarbital sodium (80 mg/ml) and perfused with ice-cold 0.9% saline, finally embedded in OCT (SAKURA#4583). Samples were frozen by liquid nitrogen & isopentane for 45 s and then cut into 8 μm slices. The slices fixed and permeabilized by acetone for 5 min, followed by blocking with 5% donkey serum in PBS with 0.2% BSA for 1 h at room temperature. Incubate with primary antibody (VDAC1, ab14734, abcam, 1:250) in blocking solution overnight at 4 °C, then wash with PBST (0.2% Triton X-100) 3 times for 5 min. Secondary antibody (1:1000) was used for 4 h at room temperature under darkness. Images were acquired using Leica SP8 laser scanning confocal microscope equipped with a 63 NA oil objective as Z stacks, with identical imaging parameters among different phenotypes in a blinded fashion. The length and area of mitochondrion were measured by Image J (Fiji)^{51 (link)}.

Protein samples were mixed with Laemmli buffer containing β-mercaptoethanol, and proteins were separated in stain-free 4–20, 7.5%, or all kD pre-cast gels (TGX Stain-Free, BioRad, CA, United States) and electroblotted to PVDF membranes (Trans-blot Turbo Transfer pack, BioRad). Membranes were blocked in 5% milk/PBST for 60 min at r/t and incubated with primary antibodies against SYP (M0776, Dako; 1:500), HDAC2 (05–814, Millipore, 1:500), PSD-95 (610,495, BD Transduction Laboratories, 1:500), CDK-5 (05–364, Upstate, 1:2000), VDAC-1 (ab14734, Abcam; 1:4000), Vinculin (ab129002, Abcam; 1:4000), SDHA (ab14715, Abcam; 1:10000), mtCO1 (ab14705, Abcam; 1:3000), Atp5b (17247-1-AP, Proteintech, 1:5000), TOM40 (sc-11,414, Santa Cruz; 1:2000), TOM20 (11802-1-AP, Proteintech, 1:4000), OPA1 (612,606, BD Biosciences, 1,1,000), and β-tubulin (T4026, Sigma Aldrich; 1:5000) o/n at +8°C. Secondary antibodies against mouse (P0447, DAKO) and rabbit (P0399, DAKO), diluted 1:5000 in 1% milk/PBST +0.01% SDS, were incubated for 60 min at r/t. Antibody detection and signal intensity quantification was performed using the ChemiDoc XRS+ imaging system (BioRad, CA, United States) utilizing stain-free technology for total protein normalization, or the Odyssey Infrared Imaging system (LI-COR Biosciences).

Western blot was performed as previous described^{31 (link)}. The primary antibodies used were as follows: anti-UCP2 (sc-6526, Santa Cruz), anti-PPARα (ab24509, Abcam, Cambridge, MA, US), anti-CPT1α (ab128568, Abcam), anti-fibronectin (F3648, Sigma Aldrich), anti-collagen I (1310–01, Southern Biotech), anti-HIF-1α (ab1, Abcam), anti-PHD2 (4835, Cell Signaling Technology), anti-VDAC (ab14734, Abcam), and anti-Tubulin (T6074, Sigma Aldrich). Western blot were performed at least three times independently. Quantification was performed by measurement of the intensity of the signals with the aid of National Institutes of Health Image software package.

Lysates from the above mentioned assays were analyzed by SDS-PAGE, using standard 12% SDS-PAGE to resolve mCherry and its XP-fusion variants, and precast Novex™ 10–20% tricine protein gels (Thermo Fisher) to resolve XPs and enterovirus 2B. Proteins were then transferred to 0.2 µm nitrocellulose membranes and blocked with 4% Marvel milk powder in phosphate-buffered saline (PBS). Immunoblotting of mCherry was performed using anti-mCherry antibody (Abcam, ab167453, 1:3000). A custom rabbit polyclonal antibody raised against XP peptide SNSGNRVSQDQNLQ (GenScript; only able to detect strongly overexpressed XP, 1:250) and an anti-Strep mouse antibody (Abcam, ab184224, 1:1000) were used for detecting HAstV1 XP and Strep-tagged proteins, respectively. The following antibodies were used for cellular targets: anti-tubulin (Abcam, ab15568, 1:500), anti-VDAC1 (Abcam, ab14734, 1:1000), and anti-LAMIN A + C (Abcam, ab133256, 1:3000). Immunoblots were imaged on a LI-COR ODYSSEY CLx imager and analyzed using Image Studio™ version 5.2.