Journal: iScience

Article Title: BCL2L13 at endoplasmic reticulum-mitochondria contact sites regulates calcium homeostasis to maintain skeletal muscle function

doi: 10.1016/j.isci.2024.110510

Figure Lengend Snippet:

Article Snippet: After blocking, primary antibodies were incubated overnight at 4°C: mouse anti-MYH2 (1:500, A4.74, Santa Cruz Biotechnology), mouse anti-ATP5a (1:500, ab14748, Abcam, Waltham, MA, United States), mouse anti-PDHα (1:1000, ab110333, Abcam), rabbit anti-PDH E1α phospho-serine 293 (1:1000, ab177461, Abcam), rabbit anti-BCL2L13 (1:500, N-ter, HPA050377, Sigma), rabbit anti-BCL2L13 (1:500, center, HPA030994, Sigma), mouse anti-FACL4 (1:500, ab155282, Abcam), mouse anti-NDUFS3 (1:1000, ab14711, Abcam), mouse anti-SERCA2 (1:500, ab150435, Abcam), rabbit anti-RyR1 antibody (Marks’ lab, Columbia University, NY, USA, Cat. #: 5,029, Aa 1327–1339), rabbit anti-MCU (1:1000, HPA016480, Sigma), mouse anti-SERCA1 (1:1000, ab2819, Abcam), mouse anti-αTubulin (1:2000, T6199, Sigma), mouse anti-IP3R-I/II/III (1:200, sc-377518, Santa Cruz biotechnology) and rabbit anti-cleaved Caspase 3 (1:500, 9661S, Cell signaling, Danvers, United States).

Techniques: Virus, Subcloning, Recombinant, Staining, Labeling, Gel Extraction, SYBR Green Assay, Reverse Transcription, Western Blot, esiRNA, Fluorescence In Situ Hybridization, Clone Assay, Plasmid Preparation, Software, Microscopy, Mass Spectrometry, Transmission Assay, Imaging

Journal: Genetics

Article Title: Cross-Talk Between Mitochondrial Fusion and the Hippo Pathway in Controlling Cell Proliferation During Drosophila Development

doi: 10.1534/genetics.115.186445

Figure Lengend Snippet: ChChd3 localizes to the inner mitochondrial membrane and is required for mitochondrial fusion and crista formation. (A) Schematic diagram of Drosophila ChChd3 protein domain structure and sequence comparison of Drosophila ChChd3 with human ChChd3 and mouse ChChd3 within the CHCH domain. (B–B′′′) S2 cells showing colocalization of ChChd3 and ATP5A. Samples were stained with anti-ChChd3 and anti-ATP5A antibodies. (C) Western blot analysis of cytosolic and mitochondrial fractions separated by centrifugation. Samples were probed with anti-ChChd3, anti-ATP5A (inner membrane), and anti-tubulin antibodies. ChChd3 is enriched in the mitochondrial fraction. (D) Western blot analysis of ATP5A (detected by anti-Total OXPHOS), ChChd3, and Tom20 proteins from S2 cell mitochondria. Isolated mitochondria were treated with the indicated concentrations of digitonin followed by Proteinase K digestion. (E–F′′′) Wing imaginal discs containing control (E–E′′′) or ChChd3D1 homozygous mutant (F–F′′′) clones stained with anti-β-Gal and anti-ATP5A antibodies. Clones are marked by the absence of β-Gal and the dashed white line. ChChd3 mutant clone cells display punctate ATP5A staining. (G) Larval body wall cells from the control larvae expressing UAS-mito-GFP with Mef2-Gal4 and showing mitochondria with tubular morphology. (H) Knockdown of ChChd3 in the larval body wall cells results in shorter mitochondria. (I and J) TEM images of mitochondria from wild-type (I) or ChChd3D1 mutant larvae. Crista content was reduced in ChChd3D1 mutant mitochondria. (K and L) TEM images of adult indirect flight muscle from the Mhc-Gal4 control (K) and ChChd3 knockdown flies (L). Knockdown of ChChd3 leads to fragmented mitochondria and reduced crista content. Bars, 10 μm in B; 40 μm in E; 10 μm in G; 0.1 μm in I; and 2 μm in K.

Article Snippet: We used the following primary antibodies: chicken anti-GFP (1:2000; Abcam), mouse anti-RFP (1:2000; Abcam), mouse anti-β-galactosidase (1:2000; Abcam), mouse anti-armadillo (1:100; DHSB N2 7A1), rabbit anti-cleaved Caspase 3 (1:100; Cell Signaling), rabbit anti-phosphohistone 3 (Ser10) (1:500; Millipore, Bedford, MA), and mouse anti-ATP synthase-α (anti-ATP5A; 1:500; Mitosciences).

Techniques: Sequencing, Staining, Western Blot, Centrifugation, Isolation, Mutagenesis, Clone Assay, Expressing

Journal: bioRxiv

Article Title: Differential labelling of human sub-cellular compartments with fluorescent dye esters and expansion microscopy

doi: 10.1101/2023.02.21.529394

Figure Lengend Snippet: By implementing NHS esters alongside antibodies it is possible to get context for those specific protein targets in relation to the broader cellular architecture. (A) 4x EExM images of NHS Alexa488 (cyan) alongside antibody labels for cis-golgi marker GM130 (yellow) and the mitochondrial ATP5A1 ATP synthase (magenta). (B) Gallery of 4x EExM images showing examples of the preferential compartment labelling by esters (all applied pre-gelation) alongside immunolabelled compartments, including (i) the endoplasmic reticulum network (as labelled with antibodies recognising KDEL in the lumen) alongside NHS AZ488, (ii) the mitochondria as shown by immunolabelling for ATP5A1, alongside NHS ATTO647N, and (iii) the golgi, targeted with GM130 antibodies, alongside NHS BODIPY493/503. Scale bars (expansion factor rescaled): (A) 3.75 μm; (Bi) 2.5 μm, (Bii) 2.5 μm, (Biii) 5 μm.

Article Snippet: The primary antibodies used in this study were rabbit polyclonal KDEL (Thermo Fisher Scientific, code: PA1-013), rabbit monoclonal GM130 (abcam, code: ab52649) and mouse monoclonal ATP5A1 (Thermo Fisher Scientific, code: 43-9800).

Techniques: Marker