Journal: medRxiv

Article Title: Inhibition of SHP2 ameliorates psoriasis by decreasing TLR7 endosome localization

doi: 10.1101/2020.09.28.20202861

Figure Lengend Snippet: (A) Expression of PTPN11 ( gene encoding SHP2 ) in skin lesions in psoriatic patients compared with skin from healthy donors based on microarray data (No. GSE14905). (B) Expression of PTPN11 in human PBMCs from psoriatic patients (n=14) and normal controls (n=16). (C) Western blot analysis of PBMCs lysates derived from psoriatic patients and normal controls. (D) Representative SHP2 staining in skin sections from psoriatic patients (n=13) and normal controls (n=5). Scale bars: 200 μm. (E) The catalytic activity of SHP2 was measured in human PBMCs lysates derived from psoriatic patients (n=25) and normal controls (n=25). (F) Representative p-ERK staining of skin sections from psoriatic patients and normal controls. Scale bars: 200 μm. (G) Quantitative PCR analysis of Ptpn11 mRNA levels in the IMQ-treated or non-treated dorsal back from C57BL/6J mice at day 5 (n=6/group). Data were normalized to GAPDH expression. (H) Representative histological sections of IMQ-treated or non-treated dorsal back from C57BL/6J mice at day 5. Scale bar: 100 μm. Data represent mean ± SEM. P values are determined by Two-tailed Mann-Whitney U test (A and B) or Two-tailed Student’s t test (E and G). * P <0.05, ** P <0.01.

Article Snippet: For immunohistochemistry, the human and mouse skin paraffin sections were deparaffinized, rehydrated, and antibody retrieved with sodium citrate, blocked, then stained with anti-SHP2 (Santa Cruz, catalog sc-7384), anti-ERK (Cell Signal Technology, catalog 4695), anti-CD68 (Cell Signal Technology, catalog 76437), anti-p-p65 (Cell Signal Technology, catalog 3033), anti-Ki67 (Abcam, catalog ab15580) were used at 1:100 overnight at 4°C.

Techniques: Expressing, Microarray, Western Blot, Derivative Assay, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY

Journal: Science (New York, N.Y.)

Article Title: Interpretation of cancer mutations using a multi-scale map of protein systems

doi: 10.1126/science.abf3067

Figure Lengend Snippet: (A) Interactions defining the PIK3CA-actomyosin complex and the context of this system in the NeST hierarchy. (B) Mutation frequencies of genes in this system (shown top seven frequently mutated genes in each cohort). (C) Signal (spectral count fold change versus control) of actomyosin proteins (MYH9, MYH10, MYO1C, LIMA1) in AP-MS experiments with cancer protein baits. Red dots represent signal as interactors of PIK3CA in the CAL-33 cell line; gray dots show corresponding data for other cancer proteins. (D) Per-cell counts of proximity ligation foci when probing for PIK3CA and MYH9 in CAL-33 cells; significantly more foci are observed when probing for both proteins. ***: P <0.001 by one-tailed Student’s T test. (E) CAL-33 cell lines expressing FLAG-PIK3CA (as used in AP/MS experiments) were probed with anti-FLAG and anti-MYH9 antibodies and imaged by DNA-PAINT. Substantial colocalization is observed in small, membrane-proximal puncta. Representative subfields are shown below; scale bar is 500nm. (F) CAL-33 cells were treated with DMSO, blebbistatin (10 μM) and/or alpelisib (0.5 μM) and harvested with indicated antibodies for immunoblotting (N = 3; a representative image is displayed). Signals were normalized relative to total protein and loading control. Error bars indicate mean ± standard error; *: P < 0.05 by one-way ANOVA; images are representative of 3 independent experiments; N = 6 for all measurements. (G) Comparison of the RPPA-measured abundance of indicated phosphorylated/total proteins between cancer cell lines (78) (N = 899) with MYH9 (N = 108) or MYH10 (N = 82) mutations and the wild-type lines. (H) Genomic alterations of PIK3CA and MYH9 in the TCGA-HNSC cohort show a pattern of mutual exclusivity. Wilcoxon rank sum test was used for G and H. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

Article Snippet: The different combinations of primary antibodies used were: anti-PIK3CA (Invitrogen, Cat# MA5–17149) diluted at 1/400; anti-FLAG (Cell Signaling Technologies, Cat# 14793S) diluted at 1/800, and anti-MYH9 (ThermoFisher, Cat#PA-29673), diluted at 1/500.

Techniques: Mutagenesis, Ligation, One-tailed Test, Expressing, Western Blot

Journal: World Journal of Gastroenterology

Article Title: To screen or not to screen? Celiac antibodies in liver diseases

doi: 10.3748/wjg.v23.i5.776

Figure Lengend Snippet: Research on screening celiac disease in patients with primary biliar cholangitis

Article Snippet: According to Bizzaro et al[ ], 26.7% of PBC patients exhibited tTG positivity on performing at least one of six different ELISA tests (human recombinant, Eurospital; human recombinant, Pharmacia; human placenta, Euroimmun; human red blood cells, Inova; guinea pig liver, Eurospital; and guinea pig liver, Inova); however, a true association between PBC and CD was present in only 2% of patients who exhibited EmA positivity and showed histological patterns indicative of CD.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: World Journal of Gastroenterology

Article Title: To screen or not to screen? Celiac antibodies in liver diseases

doi: 10.3748/wjg.v23.i5.776

Figure Lengend Snippet: Research screening celiac disease in patients with autoimmune hepatitis

Article Snippet: According to Bizzaro et al[ ], 26.7% of PBC patients exhibited tTG positivity on performing at least one of six different ELISA tests (human recombinant, Eurospital; human recombinant, Pharmacia; human placenta, Euroimmun; human red blood cells, Inova; guinea pig liver, Eurospital; and guinea pig liver, Inova); however, a true association between PBC and CD was present in only 2% of patients who exhibited EmA positivity and showed histological patterns indicative of CD.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The ALS- and FTD-associated proteins Annexin A11 and CHMP2B act sequentially in membrane repair

doi: 10.1101/2024.11.19.624330

Figure Lengend Snippet: A . Schematic representation of YFP-CHMP2A deletions (described in ). B . Resolved lysates from 293T cells transfected with vectors encoding GFP, or the constructs from A were examined by western blotting with antisera raised against GFP or p42/p44 MAPK. C , D . Representative images of CAL-51 cells transiently transfected with GFP or the constructs depicted in (A) and imaged live. Scale bars represent 5 μm. E, F . Quantification of nucleocytoplasmic ratio of YFP fluorescence from CAL-51 cells transfected with GFP or the constructs described in A. Graphs show mean ± S.E.M. from n = 15-51 cells per condition across N = 1 independent experiment with significance calculated by two tailed T-test. Error bars below zero cannot be displayed on a logarithmic y-axis. G . Sequence alignment of α2 helices in mammalian ESCRT-III proteins. A cluster of basic residues at the N-terminus of these helices highlighted in red.

Article Snippet: Anti-GFP (clone 7.1/13.1) was from Roche; in-house anti-CHMP2B (3335) was developed by BioGenes as previously described ; anti-GAPDH (clone 6C5, MAB374) was from Millipore; anti-ALIX (12-422-1-AP), anti-ANXA7 (10154-2-AP), anti-ANXA11 (10479-2-AP) and anti-ALG-2 (12303-1-AP) were from Proteintech; anti-PfO (ab225685) was from Abcam; anti-p42/p44 MAPK (9102) was from Cell Signalling Technology; anti-HIS (652502, clone J099B12) was from Biolegend.

Techniques: Transfection, Construct, Western Blot, Fluorescence, Two Tailed Test, Sequencing

Journal: Leukemia

Article Title: Discriminating activities of DEAD-Box Helicase 41 from myeloid malignancy-associated germline variants by genetic rescue

doi: 10.1038/s41375-022-01753-4

Figure Lengend Snippet: Key Resource Table

Article Snippet: Reagent Source Identifier Bacterial strains JM109 competent cells Promega L2001 Experimental model: Cell lines Mouse: hi- Ddx41 +/+ and hi- Ddx41 +/− cells This paper N/A CRISPR/Cas-9 system Predesigned Alt-R CRISPR-Cas9 gRNA for mouse Ddx41 Integrated DNA Technologies Mm.Cas9.DDX41.1.AA, Mm.Cas9.DDX41.1.AB, Mm.Cas9.DDX41.1.AC sNLS-spCas9-sNLS Aldevron 9212 Antibodies Flag-tag rabbit mAb Cell Signaling Technology 14793 Alexa Fluor 594 chicken anti-rabbit IgG (H+L) Invitrogen A21442 DDX41 mouse mAb Novus Biologicals H00051428-M01 DDX41 rabbit pAb Cocalico Biologicals, INC. Bresnick laboratory-generated β-actin mouse mAb Cell Signaling Technology 3700S GAPDH rabbit mAb Cell Signaling Technology 2118 Recombinant DNA pMSCV-PIG Mitchell Weiss (St. Jude Children’s Research Hospital) N/A pMSCV-PIG-DDX41 WT This paper N/A pMSCV-PIG-DDX41-G173R pMSCV-PIG-DDX41-R293H pMSCV-PIG-DDX41-K331del pMSCV-PIG-DDX41-R525H This paper N/A Chemicals Pierce TM ECL 2 Western Blotting Substrate Thermo Fisher Scientific 80196 SuperSignal TM West Femto Maximum Sensitivity Substrate Thermo Fisher Scientific 34096 Power SYBR Green Master Mix Applied Biosystems 4367659 TRIzol Reagent Thermo Fisher Scientific 15596 Phusion High-Fidelity DNA Polymerase Thermo Fisher Scientific F530S P3 Primary Cell 4D-Nucleofector ® X Kit Lonza V4XP-3032 Critical commercial kit RNAeasy Micro Kit Qiagen 74004 QIAquick Gel Extraction Kit Qiagen 28706 QIAquick PCR Purification Kit Qiagen 28106 Software Image Studio Lite (version 5.2) Image studio acquisition software https://www.licor.com/ Prism 9 GraphPad https://www.graphpad.com/ SnapGene viewer Dotmatics https://www.snapgene.com/snapgene-viewer/ Open in a separate window Key Resource Table.

Techniques: CRISPR, Recombinant, Western Blot, SYBR Green Assay, Gel Extraction, Purification, Software

Journal: Animal Models and Experimental Medicine

Article Title: Elimination of GGTA1 , CMAH , β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions

doi: 10.1002/ame2.12461

Figure Lengend Snippet: Construction and evaluation of 4KO piglets in xenotransplantation. (A) Schematic diagram of method for generating 4KO piglets. (B) Schematic of the 10 modified alleles. We generated the 4KO edits using CRISPR–Cas9 with gRNAs targeting the 2 copies of GGTA1 , 2 copies of CMAH , 4 copies of β4GalNT2 and 2 copies of the CIITA . (C) Deficiency of GGTA1 , CMAH , β4GalNT2 , and CIITA can impair the antigens of αGal, Neu5Gc, SDa, and SLA class II molecules, respectively. Flow cytometry was used to verify the deficiency of αGal, Neu5Gc, SDa, and SLA class II‐DR molecules at the cellular level in 4KO piglets. PBMCs of 4KO pigs had negative population cells (WT = 3, 4KO = 3). (D) Immunofluorescence results showed that αGal, Neu5Gc, SDa, and SLA class II molecules were not expressed in kidney tissue of 4KO piglets, and GGTA1 , CMAH , β4GalNT2 , and CIITA are genes were used to knockout the antigen of αGal, Neu5Gc, SDa and SLA class II molecules, respectively. Scale bar: 20 μm. (E) Verification by HE staining of the viability of 4KO tissue after gene editing; Scale bar: 100 μm.

Article Snippet: Stimulated pig cell samples were 2 × 10 5 WT or 4KO pig PBMCs treated with 50 μg/mL Mitomycin C (S8146, Selleck, Houston, TX, USA) for 20 min at 37°C to block their capacity for proliferation.

Techniques: Modification, Generated, CRISPR, Flow Cytometry, Immunofluorescence, Knock-Out, Staining

Journal: Animal Models and Experimental Medicine

Article Title: Elimination of GGTA1 , CMAH , β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions

doi: 10.1002/ame2.12461

Figure Lengend Snippet: Changes in the pig immune system induced by CIITA deficiency (WT = 3, 4KO = 3): (A) Percentage of the population of CD3 in WT and 4KO. (B) Percentage of the population of γδT in WT and 4KO. (C) 4KO CD3 T cells are not significantly different from WT. (D) 4KO γδT are not significantly different from WT. (E) Percentage of the population of CD4+ T and CD8+ T in WT and 4KO. (F) 4KO CD4+ T cells significantly decreased relative to WT; 4KO CD8+ T cells significantly increased relative to WT. (G) B cell population of WT and 4KO PBMCs.( n = 3). (H) No significant difference between WT and 4KO in the population of B cells. (I) Percentage of 4KO NK cell population significantly decreased relative to WT. (J) NK cell population of WT and 4KO PBMCs. ** p < 0.01; * p < 0.05.

Article Snippet: Stimulated pig cell samples were 2 × 10 5 WT or 4KO pig PBMCs treated with 50 μg/mL Mitomycin C (S8146, Selleck, Houston, TX, USA) for 20 min at 37°C to block their capacity for proliferation.

Techniques:

Journal: Animal Models and Experimental Medicine

Article Title: Elimination of GGTA1 , CMAH , β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions

doi: 10.1002/ame2.12461

Figure Lengend Snippet: (A) Verifying the function of CIITA deficiency, T cell proliferation was analyzed based on dilution of the proliferation dye CSFE with each cell division, Proliferation of human CD4+ T cells is depicted as percent of divided cell (WT = 3; 4KO = 3). (B) 4KO has lower immunogenicity to lead human CD4+ T proliferation than WT (WT = 3; 4KO = 3). (C) Verifying the Serum‐mediated antibody binding reaction of WT and 4KO, compared with WT, 4KO show significantly reduced binding to human IgG (a) and IgM (WT = 8; 4KO = 8). (D) Verifying the Human complement‐dependent cytotoxicity of WT and 4KO, 4KO show significantly lower antibody‐dependent complement cytotoxicity compared with WT (WT = 5; 4KO = 5). Unpaired two‐tailed t ‐test; ** p < 0.01; * p < 0.05).

Article Snippet: Stimulated pig cell samples were 2 × 10 5 WT or 4KO pig PBMCs treated with 50 μg/mL Mitomycin C (S8146, Selleck, Houston, TX, USA) for 20 min at 37°C to block their capacity for proliferation.

Techniques: Immunopeptidomics, Binding Assay, Two Tailed Test

Journal: Science Advances

Article Title: Expression of expanded GGC repeats within NOTCH2NLC causes behavioral deficits and neurodegeneration in a mouse model of neuronal intranuclear inclusion disease

doi: 10.1126/sciadv.add6391

Figure Lengend Snippet: ( A and B ) Immunofluorescence against NeuN in the hippocampus (A) and calbindin in the cerebellum (B) from NOTCH2NLC mice. Red, NeuN; green, calbindin. Scale bars, 100 μm. ( C and D ) Quantification of the staining intensity of NeuN (C) and the number of Purkinje neurons (D). Data are presented as means ± SEM. [ N = 3 mice per group, * P = 0.0152 (C), ** P = 0.0086 (D), two-tailed t test]. ( E ) Nissl staining of the cortex and cerebellum from NOTCH2NLC-(GGC) 17 and NOTCH2NLC-(GGC) 98 mice at P50. Red arrowheads indicate dark shrunken neurons; red arrows indicate the Purkinje neurons. Scale bar, 50 μm. ( F and G ) Quantification of the number of Purkinje neurons in the cerebellum (F) and the number of normal neurons in the cortex (G). Data are presented as means ± SEM. [ N = 3 mice per group, *** P = 0.0001 (F) and ** P = 0.0033 (G), two-tailed t test]. ( H and I ) Western blotting against NeuN in the hippocampus (HIP) and against calbindin in the cerebellum (CB) from NOTCH2NLC-(GGC) 17 and NOTCH2NLC - (GGC) 98 mice at P50. Normalized relative expressions of NeuN and calbindin are present in (I). Data are presented as means ± SEM. ( N = 3 mice per group, * P = 0.0434 and ** P = 0.0038, two-tailed t test). ( J and K ) Immunofluorescence against GFAP and Iba1 in the HIP and CB from NOTCH2NLC - (GGC) 17 and NOTCH2NLC - (GGC) 98 mice at P50 (J). (K) Quantification of GFAP and Iba1 relative staining intensity. Data are presented as mean ± SEM. [ N = 3 mice per group, **** P < 0.0001 (GFAP in HIP), ** P = 0.0055 (GFAP in CB), ** P = 0.0023 (Iba1 in HIP), and ** P = 0.0048 (Iba1 in CB), two-tailed t test]. Green, GFAP; red, Iba1. Scale bar, 100 μm.

Article Snippet: Primary antibodies used in this study include the following: Flag (Sigma-Aldrich, F1804), HA (Cell Signaling Technology, 3724S), Myc (Cell Signaling Technology, 2276S), calbindin (Sigma-Aldrich, C9848), vinculin (Sigma-Aldrich, V9131), NeuN (Cell Signaling Technology, 24307s), GFAP (Cell Signaling Technology, 3670S), IBA1 (Wako, 019-19741), ubiquitin (Sigma-Aldrich, 05-1307), P62 (BD Biosciences, 610832), GFP (Invitrogen, A-11122), β-actin (Cell Signaling Technology, 3700S), Tuj1 (Abcam, ab18207), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, 60004-1-Ig), NOTCH2NL (Gentex, GTX117659), and hnRNPM (Santa Cruz Biotechnology, sc-20002).

Techniques: Immunofluorescence, Staining, Two Tailed Test, Western Blot