Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Novel Tmem173 allele reveals importance of STING N-terminus in trafficking and type I IFN production

doi: 10.4049/jimmunol.1501415

Figure Lengend Snippet: A) Haplotype assortment of IFNβ by F2 mice at marker D18Mit202 in the vicinity of Tmem173. B) Identified polymorphisms in MOLF STING change predicted motifs and structure of the protein; (C) A reporter assay with B6 or MOLF STING cDNA in pEF-Bos activating Ifnb1 promoter in response to different STING agonists; (D) Macrophages were transfected (Lipofectamine) with STING agonists or pI:C for 16 hrs, and IFNβ was determined by ELISA; (E) STING KO MEFs stably reconstituted with B6 (left) or MOLF (right) mCherry-STING were stimulated for 2h with lipofectamine alone (media) or 2′3′cGAMP (4ug/mL); Cells were fixed and permeablized with 4% PFA and MeOH, co-stained with ER marker - Grp94 (Green).

Article Snippet: Antibodies: (Cell Signaling): STING #3337; p65 #8242; Phospho-p65 #3033; ERK #4695; Phospho -ERK #4370; p38 #9212; Phospho -p38 #4511; TBK1 #3013; Phospho -TBK1 #5483; Phospho -IRF3 #4947; JNK #9258; Phospho -JNK #9251; HA-Tag #3724. (ENZO): Grp94, #ADI-SPA-850.

Techniques: Marker, Reporter Assay, Transfection, Enzyme-linked Immunosorbent Assay, Stable Transfection, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Novel Tmem173 allele reveals importance of STING N-terminus in trafficking and type I IFN production

doi: 10.4049/jimmunol.1501415

Figure Lengend Snippet: A) 293T cells were co-transfected for 16 hrs with reporters (driven by different DNA-binding elements of Ifnb1 promoter) and 50 ng of plasmid DNA encoding individual SNPs from MOLF followed by luciferase measurements. (B) 293T cells were transfected similar to (A) with 0.5 ng of plasmid DNA and luciferase was measured 16 hrs after activation of cells with 4 μg/ml of c-di-AMP. (C) STING KO MEFs stably reconstituted with L47V-A48G, or S53L single-mutants of mCherry-STING were stimulated for 2h with lipofectamine alone or with c-di-AMP (4ug/mL), permeablized with 4% PFA and MeOH, co-stained for ER with Grp94 (Green). (D) The lysates from cells in (A) were analyzed by western blot hybridization..

Article Snippet: Antibodies: (Cell Signaling): STING #3337; p65 #8242; Phospho-p65 #3033; ERK #4695; Phospho -ERK #4370; p38 #9212; Phospho -p38 #4511; TBK1 #3013; Phospho -TBK1 #5483; Phospho -IRF3 #4947; JNK #9258; Phospho -JNK #9251; HA-Tag #3724. (ENZO): Grp94, #ADI-SPA-850.

Techniques: Transfection, Binding Assay, Plasmid Preparation, Luciferase, Activation Assay, Stable Transfection, Staining, Western Blot, Hybridization

Journal: Human Molecular Genetics

Article Title: Mitochondria–lysosome membrane contacts are defective in GDAP1-related Charcot–Marie–Tooth disease

doi: 10.1093/hmg/ddaa243

Figure Lengend Snippet: GDAP1 interacts with SYNTAXIN 17 (STX17) and LC3 in MAMs. ( A ) Negative interactions by co-IP assays of GDAP1 with DRP1 (mitochondrial fission in MAMs), ACSL1 (fatty acid metabolism in MAMs), GRP75 (Ca 2+ channel in MAMs), BECLIN-1 (ER-mitochondria tethering and autophagosome formation), ATG4 (unique redox sensor essential for maturation of autophagosomes) and RAB7 (trafficking, maturation, and fusion of endocytic and autophagic vesicles). ( B and C ) Co-IP assay of endogenous GDAP1 and STX17 (B) or LC3 (C) in SH-SY5Y cells. ( D and E ) Representative images of the interaction between GDAP1 and STX17 (D) or LC3 (E) in SH-SY5Y cells by PLA from 3 independent experiments. Scale bar: 10 μm. ( F ) Western blot of subcellular fractions from SH-SY5Y and G4 cells and quantification of relative protein levels in MAMs fraction (three or four independent experiments). C, cytosol; ER, endoplasmic reticulum; pM, pure mitochondria; MAM, mitochondria associated membranes. Data information: In (F), data represent mean ± SD and individual values are displayed as dots. ANOVA followed by Sidak’s post hoc test.

Article Snippet: The following antibodies were used: ACSL1 rabbit antibody (Cell Signaling, #4047), ATG4B mouse antibody (MBL, M134-3), ATG4B rabbit antibody (Abcam, ab154843), BECLIN-1 rabbit antibody (Cell Signaling #3738), β-ACTIN mouse antibody (Sigma-Aldrich, A5316), TUBB3 rabbit antibody (Sigma-Aldrich, T2200), TUBB3 mouse antibody (Promega, G7121), DRP1 mouse antibody (BD Biosciences, 611113), FACL-4 mouse antibody (Santa Cruz, sc-365230), GDAP1 rabbit antibody (Sigma-Aldrich, HPA024334), GDAP1 mouse antibody (Abcam, ab194493), GRP75 rabbit antibody (Santa Cruz, sc-13967), HA-probe mouse antibody (Santa Cruz, sc-7392), LAMP-1 rabbit antibody (Sigma-Aldrich, L1418 and Abcam ab24170), LAMP-1 mouse antibody (DSHB, H4A3), LC3 mouse antibody (LifeSpan Biosciences, LS-C165694), LC3B rabbit antibody (Sigma-Aldrich, L7543), MITOFUSIN-2 rabbit antibody (Sigma-Aldrich, M6319), PIK fyve mouse antibody (Santa Cruz, sc-100408), p62/SQSTM1 rabbit antibody (Sigma-Aldrich, P0067), RAB7 mouse antibody (Sigma-Aldrich, R8779), SYNTAXIN 17 rabbit antibody (Sigma-Aldrich, HPA001204), TFEB rabbit antibody (LifeSpan Biosciences, LS-C353036), TOM20 mouse antibody (BD Biosciences, 612278), V5 rabbit antibody (Sigma-Aldrich,V8137) and Alexa fluorophore-conjugated secondary antibodies from Molecular Probes (Invitrogen).

Techniques: Co-Immunoprecipitation Assay, Western Blot

Journal: Immunity

Article Title: Loss of Neurological Disease HSAN-I-Associated Gene SPTLC2 Impairs CD8 + T Cell Responses to Infection by Inhibiting T Cell Metabolic Fitness

doi: 10.1016/j.immuni.2019.03.005

Figure Lengend Snippet: (A) Antigen-specific P14 CD8+ T cells were purified on day 6 (D6) after LCMV-Armstrong infection from the “P14 chimeric mice”. Naïve P14 T cells were also purified before LCMV infection (D0) to analyze the basal level of SPTLC2 protein by western blot. Sptlc2-deficient P14 CD8+ T cells were included as negative controls. Bar graphs show the densitometry quantification of the SPTLC2 immunoblot bands. GRP94 was used as a loading control. (B) P14 TCR transgenic mouse splenocytes were stimulated with GP33–41 peptide or various cytokines as indicated for 3 days for immunoblot. Each lane represents an individual mouse sample (A-B). (C-F) Sptlc2Flox/FloxCd4-cre (Fl/Fl) mice and wildtype littermates (+/+) were infected with LCMV-Armstrong and sacrificed 8 days later. Representative FACS plots and bar graphs (C-D) show the percentages and numbers of DbGP33–41 and DbNP396–404 tetramer-positive splenic Sptlc2-deficient or wildtype CD8+ T cells (C), the effector cytokine-producing CD8+ T cells after restimulation with or without the LCMV peptide GP33–41 for 6 hours (D) and viral titers in mouse serum (E) and spleens (F). Data are expressed as mean ± SD (error bars) and are representative of two (six (A) or three (B) pairs of mice in total) or three (C-F, eight pairs of mice in total) independent experiments (three in each experiment). *p<0.05; **p<0.01. See also Figure S2.

Article Snippet: GRP94 antibody , Cell Signaling Technology , Cell Signaling Technology Cat# 20292,.

Techniques: Purification, Infection, Western Blot, Control, Transgenic Assay

Journal: Immunity

Article Title: Loss of Neurological Disease HSAN-I-Associated Gene SPTLC2 Impairs CD8 + T Cell Responses to Infection by Inhibiting T Cell Metabolic Fitness

doi: 10.1016/j.immuni.2019.03.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: GRP94 antibody , Cell Signaling Technology , Cell Signaling Technology Cat# 20292,.

Techniques: Purification, Recombinant, Western Blot, Membrane, Software