Journal: Immunity

Article Title: Signaling through the inhibitory Fc receptor FcγRIIB induces CD8 T cell apoptosis to limit T cell immunity

doi: 10.1016/j.immuni.2019.12.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Dr. Mark Cragg InVivoMAb anti-mouse CD16/32 (clone 2.4G2) BioXcell Cat# BE0307, RRID:AB_2736987 Bacterial and Virus Strains Biological Samples Emory Transplant Center Biorepository Emory IRB protocol #00046593 Chemicals, Peptides, and Recombinant Proteins GolgiPlug BD Biosciences Cat# 555029 OVA peptide 257–264 (SIINFEKL) GenScript Cat# RP10611 CTLA-4Ig (abatacept) Bristol-Myers Squibb, Abatacept (Orencia) Anti-CD28 domain antibodies Bristol-Myers Squibb Collagenase (type 1A) Sigma-Aldrich C2654 Hyaluronidase (type 1) Sigma-Aldrich H3506 Collagenase P Sigma-Aldrich Ref# 11213865001 Collagenase D Sigma-Aldrich Ref# 1088866001 Recombinant Fgl2 R&D Systems R&D Cat# 5257-FL-050 Critical Commercial Assays MACS CD8a+ T cell isolation kit, mouse Miltenyi Biotec 130-104-075 Quick-RNA MicroPrep Kit Zyma Research R1051 SMART-seq v4 cDNA synthesis kit Takara Cat# 634894 NexteraXT kit Illumina FC-131–1096 Mouse Fgl2 ELISA Biolegend Biolegend Cat# 437807 RNeasy Plus Micro Kit Qiagen Qiagen Cat# 74034 High Capacity cDNA reverse transcription kit ThermoFisher ThermoFisher Cat# 4368814 HT HG-U133 Plus PM BeadChip Affymetrix Thermo Fisher Cat# 901261 Lightning Link R-PE Antibody Labeling kit Novus Biologicals Cat # 703–0010 CountBright Beads Life Technologies Cat # C36950 Deposited Data RNAseq data NCBI Gene Expression Omnibus (GEO) GSE118439 Experimental Models: Cell Lines B16 Melanoma-OVA Brown, et al., 2001 , provided by Dr. Yang-Xin Fu Experimental Models: Organisms/Strains NCI C57Bl/6Ncr (NCI grantee program) Charles River Stock #556 NCI B6-LY5.1/Cr (NCI grantee program) Charles River Stock #564 OT-I Hogquist et al., 1994 OT-II Barnden et al., 1998 mOVA (C57Bl/6 background, H-2b) Ehst et al., 2013 Dr. Marc Jenkins B6.129P2-Aicda tm1(cre)Mnz /J ( Aicda −/− ) The Jackson Laboratory Stock #007770 B6;129S-Fcgr2btm1Ttk/J ( Fcgr2b −/− ) The Jackson Laboratory Stock #002848 EM:06078 Fcgr2b Fcgr2bB6null B6(Cg)-Fcgr2btm12Sjv/Cnbc ( Fcgr2b −/− ) European Mutant Mouse Archive; Dr. J.S.

Techniques: Control, Virus, Recombinant, Cell Isolation, cDNA Synthesis, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Antibody Labeling, Gene Expression, Mutagenesis, Software

Journal: The FASEB Journal

Article Title: Toxoplasma gondii ROP18 I inhibits host innate immunity through cGAS‐STING signaling

doi: 10.1096/fj.202101347r

Figure Lengend Snippet: FIGURE 4 TgROP18I interacted with p62 and promoted the phosphorylation level of p62. (A, B) COS-7 cells were transfected with pcDNA3.1(+) -TgROP18I-3×Flag and/or pcDNA3.1(+) -p62-HA for 48 h, and the cell lysates were subjected to immunoprecipitation with (A) anti-Flag or (B) anti-HA antibody, and then analyzed with the indicated antibodies by WB. (C) COS-7 cells were transfected with pcDNA3.1(+) -TgROP18I-3×Flag or pcDNA3.1(+) -TgROP18I-KD-3×Flag, together with pcDNA3.1(+) -p62-HA, followed by western blot (WB) analysis. (c) Image J densitometry analysis for the level of p-p62 relative to that of p62. (D) COS-7 cells were transfected with pcDNA3.1(+) -TgROP18I-KD-3×Flag and/or pcDNA3.1(+) -p62-HA, and cell lysates were subjected to Co-IP with anti-Flag or anti-HA antibody, and analyzed by WB with the indicated antibodies. (E–F) RAW264.7 cells were infected with Toxoplasma gondii (MOI = 1) (E) RH-WT or RH-∆rop18, (F) CEP-WT or CEP-rop18I for 24 h, and the cell lysates were analyzed by WB with the indicated antibodies. Data are represented as mean ± SEM of three independent experiments. Statistical significance was assessed with One-way ANOVA. *p < .05

Article Snippet: The antibodies used for immunoblotting: rabbit monoclonal anti- β- actin, rabbit monoclonal anti- GAPDH, rabbit monoclonal anti- cGAS, rabbit monoclonal anti- STING, rabbit monoclonal anti- TBK1, rabbit monoclonal anti- pTBK1Ser172, rabbit monoclonal anti- IRF3, rabbit monoclonal anti- pIRF3- Ser396, rabbit monoclonal anti- HA- tag, rabbit monoclonal anti- pSQSTM1- Ser403 purchased from Cell Signaling Technology, USA; mouse monoclonal anti- Flagtag purchased from Sigma, Germany; rabbit monoclonal anti- SQSTM1 purchased from Bimake, USA; rabbit polyclonal anti- SAG1 raised in our lab; goat anti- rabbit IgG (H+L) secondary antibody, and goat anti- mouse IgG (H+L) secondary antibody purchased from ABclonal, China.

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Infection

Journal: The FASEB Journal

Article Title: Toxoplasma gondii ROP18 I inhibits host innate immunity through cGAS‐STING signaling

doi: 10.1096/fj.202101347r

Figure Lengend Snippet: FIGURE 5 TgROP18I inhibited IFN-γ induced recruitment of Ub, K63-Ub, K48-Ub, p62, and LC3 to the parasitophorous vacuole membrane in L929 cells. (A–E) L929 cells were stimulated with IFN-γ for 24 h (100 U/ml), and then infected with RH-WT and RH-∆rop18 strains at MOI 3 for 3 h. Representative images were shown for the parasites with SAG1 recognized in red or green by mouse monoclonal p30 antibody, and the autophagy mediators stained in red or green by (A) mouse monoclonal ubiquitin, (B) rabbit monoclonal K63-linked ubiquitin, (C) K48-linked ubiquitin, (D) p62, and (E) LC3 antibody, respectively. (a–e) The number of PVs per 25 objective fields were counted and the percentage of vacuoles positive for total (a) Ub, (b) K63-Ub, (c) K48-Ub, (d) p62, and (e) LC3 was calculated. (F–G) L929 cells were stimulated with IFN-γ for 24 h and then infected with indicated strains at MOI of 1 for 24 h. The number of parasites in each parasitophorous vacuole was determined. Data are represented as mean ± SEM of three independent experiments. Statistical significance was assessed with a two-tailed, unpaired, student's t-test. *p < .05, **p < .01, ***p < .001. Scale bar = 10 μm

Article Snippet: The antibodies used for immunoblotting: rabbit monoclonal anti- β- actin, rabbit monoclonal anti- GAPDH, rabbit monoclonal anti- cGAS, rabbit monoclonal anti- STING, rabbit monoclonal anti- TBK1, rabbit monoclonal anti- pTBK1Ser172, rabbit monoclonal anti- IRF3, rabbit monoclonal anti- pIRF3- Ser396, rabbit monoclonal anti- HA- tag, rabbit monoclonal anti- pSQSTM1- Ser403 purchased from Cell Signaling Technology, USA; mouse monoclonal anti- Flagtag purchased from Sigma, Germany; rabbit monoclonal anti- SQSTM1 purchased from Bimake, USA; rabbit polyclonal anti- SAG1 raised in our lab; goat anti- rabbit IgG (H+L) secondary antibody, and goat anti- mouse IgG (H+L) secondary antibody purchased from ABclonal, China.

Techniques: Membrane, Infection, Staining, Ubiquitin Proteomics, Two Tailed Test

Journal: The FASEB Journal

Article Title: Toxoplasma gondii ROP18 I inhibits host innate immunity through cGAS‐STING signaling

doi: 10.1096/fj.202101347r

Figure Lengend Snippet: FIGURE 6 TgROP18I alleviated IFN-γ-induced parasitophorous vacuole decoration with Ub, K63-Ub, K48-Ub, p62, and LC3 in human foreskin fibroblast (HFF) cells. (A–D) HFFs were stimulated with IFN-γ (100 U/ml) for 24 h, and then infected with the indicated T. gondii strains at MOI 3 for 3 h. Immunofluorescence was performed with mouse monoclonal p30 antibody, mouse (A) monoclonal anti-ubiquitin antibody, (B) rabbit monoclonal K63-linked ubiquitin antibody, (C) rabbit monoclonal p62 antibody (D), and rabbit monoclonal LC3 antibody. (a–d) For each staining, the number of PVs per 25 objective fields were scored, and the percentage of vacuoles that stained positive for (a) Ub, (b) K63-Ub, (c) p62, and (d) LC3 was calculated. (E–F) HFF cells were pre-stimulated with IFN-γ (100 U/ml) for 24 h and then infected with the indicated T. gondii strains at MOI of 1. Parasites per PV were measured at 24 h p.i, and the different number of tachyzoites per PV was shown as indicated. Data are represented as mean ± SEM of three independent experiments. Statistical significance was assessed with a two-tailed, unpaired, student's t-test. *p < .05, **p < .01, ***p < .001. Scale bar = 10 μm

Article Snippet: The antibodies used for immunoblotting: rabbit monoclonal anti- β- actin, rabbit monoclonal anti- GAPDH, rabbit monoclonal anti- cGAS, rabbit monoclonal anti- STING, rabbit monoclonal anti- TBK1, rabbit monoclonal anti- pTBK1Ser172, rabbit monoclonal anti- IRF3, rabbit monoclonal anti- pIRF3- Ser396, rabbit monoclonal anti- HA- tag, rabbit monoclonal anti- pSQSTM1- Ser403 purchased from Cell Signaling Technology, USA; mouse monoclonal anti- Flagtag purchased from Sigma, Germany; rabbit monoclonal anti- SQSTM1 purchased from Bimake, USA; rabbit polyclonal anti- SAG1 raised in our lab; goat anti- rabbit IgG (H+L) secondary antibody, and goat anti- mouse IgG (H+L) secondary antibody purchased from ABclonal, China.

Techniques: Infection, Immunofluorescence, Ubiquitin Proteomics, Staining, Two Tailed Test

Journal: DNA and cell biology

Article Title: Key Subdomains of Cerebral Dopamine Neurotrophic Factor Regulate Its Protective Function in 6-Hydroxydopamine-Lesioned PC12 Cells.

doi: 10.1089/dna.2023.0215

Figure Lengend Snippet: FIG. 1. Effect of 6-OHDA on the secretion and sublocalization of CDNF protein in PC12 cells. (A) CCK-8 assay showed the cell viability of PC12 cells treated with varing concentration (0, 20, 40, 60, and 80 mM) of 6-OHDA for 24 h. The value of the 0 mM group cells was set to 100%, to which the values of the other groups were normalized to. Data were presented as the mean – SD determined from analysis of three independent experiments. Comparisons between the groups were per- formed using ANOVA and Dunnett’s t-test was used as post hoc test (*p < 0.05 or **p < 0.01 vs. 0 mM group). (B) PC12 cells were transfected with Wt, M1, or M7. Lysate and medium were prepared and analyzed by immunoblotting with anti- HA antibody. (C) The value of medium versus cell lysate from Wt was set to 100%. The value of secretion of M1 and M7 was normalized to Wt. (D) Colocalization of the ER marker, calnexin, was visualized by costaining with anti-HA and anti- calnexin antibodies. Scale bar, 10 mm. (F) Colocalization of CDNF and the Golgi apparatus marker, TGN38, was visualized by co-staining with anti-HA and anti-TGN38 antibodies. Scale bar, 10 mm. (E, G) Quantitative analysis of the proportion of colocalization between CDNF and ER or Golgi. Data are presented as mean – SD determined from analysis of three independent experiments. Comparisons between the groups were performed using ANOVA and Dunnett’s t-test was used as post hoc test (*p < 0.05 or **p < 0.01 vs. Wt group). 6-OHDA, 6-hydroxydopamine; CDNF, cerebral dopamine neurotrophic factor; ER, endoplasmic reticulum; HA, hemagglutinin; M1, a1 mutation; SD, standard deviation.

Article Snippet: Hemagglutinin (HA)-Tag (6E2) Mouse mAb (2367), p44/42 ERK1/2 (9102), and phospho-p44/42 ERK1/2 (Thr202/ Tyr204) (9101), Alexa Fluor 488-conjugated goat antimouse IgG antibody (4408), and Alexa Fluor 594- conjugated goat anti-rabbit IgG antibody (8889) were obtained from Cell Signaling Technology.

Techniques: CCK-8 Assay, Concentration Assay, Transfection, Western Blot, Marker, Staining, Mutagenesis, Standard Deviation