mch climp 63  (Addgene inc)

Journal: bioRxiv

Article Title: Endoplasmic Reticulum contact sites facilitate the coordinated division of Salmonella- containing vacuole (SCV)

doi: 10.1101/2024.05.02.592158

Figure Lengend Snippet: (S2A) Percent invasion of STM WT in HeLa cells upon overexpressing mCh-EV, Sec61β-mCh, and Rtn4a-mCh (S2B) Quantitative PCR of Rtn4a upon knock down, N=3, n=3, Data is representative of mean±SD (S3C) Live cell imaging snapshots of RAW 264.7 murine macrophage cell line infected with STM WT showing ER overlap at fission site, N=1.(S2D) confocal images of infected HeLa cells showing SCV localization at cell periphery, 16h post infection. Student’s t test was used to analyse the data ****<0.0001, ***<0.001, **<0.01, *<0.05.

Article Snippet: Overexpression constructs such as: pHAGE2 mCherry-Rtn4a was a kind gift from Prof. Tom Rapoport (Addgene plasmid 86683) mCh Climp-63 and mCh sec61β was a kind gift from Prof. Gia Voeltz (Addgene plasmid 136293, Addgene 49155) , , RFP KDEL construct was a kind gift from Prof. Nagaraj Balasubramanian , GFP-LAMP1 was provided by Prof. Mahak Sharma .

Techniques: Real-time Polymerase Chain Reaction, Knockdown, Live Cell Imaging, Infection

Journal: STAR protocols

Article Title: Protocol for measuring interorganelle contact sites in primary cells using a modified proximity ligation assay.

doi: 10.1016/j.xpro.2024.102915

Figure Lengend Snippet: Figure 2. Images of PLA foci in transfected cells (A) Control PLA using IgG mouse antibody and TOMM20 (mitochondria). The PLA image demonstrates the specificity of the assay. (B) PLA for TOMM20 (mitochondria) and calnexin (endoplasmic reticulum). Boxed regions are enlarged in the bottom panels. White arrowheads show non-specific foci. Scale bar: 10 mm for the actual image and 5 mm for zoomed images. (C) Percentage of non-specific PLA foci (Calnexin-TOMM20, CLIMP63-TOMM20) in primary human fibroblast. Each point represents an individual cell. Bars show the average G SD.

Article Snippet: 1 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Mouse IgG isotype control 1:200 Invitrogen 08-6599 Fluorescent-tagged secondary antibody 1:500 Jackson ImmunoResearch 715-605-150; RRID:AB_2340862 (Anti-mouse 647), 711-165-152; RRID:AB_2307443 (Anti-Rabbit cy3) Chemicals, peptides, and recombinant proteins Dulbecco’s modified Eagle’s medium Wisent 319-005-CL Trypsin with EDTA Wisent 325-042-CL Penicillin-Streptomycin Wisent SV30010 Fetal bovine serum Corning 35-077-CV L-glutamine solution Wisent 609-065 EL 1X phosphate-buffered saline, sterile Wisent 311-425-CL Paraformaldehyde Sigma-Aldrich P6148 Bovine serum albumin Sigma-Aldrich A7906 Triton X-100 Sigma-Aldrich 9002-93-1 Epredia Immu-Mount Fisher Scientific 9990412 DAPI (40,6-diamidino-2-phenylindole, dihydrochloride) Thermo Fisher Scientific D1306 Critical commercial assays Duolink in situ green kit mouse/rabbit Sigma-Aldrich DUO92014 Duolink in situ PLA probe anti-rabbit PLUS Sigma-Aldrich DUO92002 Duolink in situ PLA probe anti-mouse MINUS Sigma-Aldrich DUO92004 Duolink in situ wash buffers, fluorescence Sigma-Aldrich DUO82049 Neon Transfection System 10 mL Kit Thermo Fisher Scientific MPK1025 Experimental models: cell lines Primary human fibroblasts (controls and DRP1 mutants) Skin biopsies (Research Ethics Board of the Children’s Hospital of Eastern Ontario (DRP1 mutants)) N/A Recombinant DNA mCherry Addgene 54517 mCherry-tagged CLIMP63 Addgene 136293 Software and algorithms ImageJ National Institutes of Health https://imagej.nih.gov/ij/ R www.r-project.org/ R4.0.5 RStudio http://www.rstudio.com/ Version 2023.03.0 + 386 Other Fisherbrand Cover Glasses Fisher Scientific 1254580 Neon transfection system Thermo Fisher Scientific 10431915 Dry bath Confocal microscope Leica Microsystems Leica SP8

Techniques: Transfection, Control