Journal: PLoS Pathogens

Article Title: Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection

doi: 10.1371/journal.ppat.1008744

Figure Lengend Snippet: Heatmap showing the significant associations by Spearman correlation between intrahepatic CXCL10, CXCR3 and HIV with liver related outcomes including fibrosis and liver enzymes (left hand panel) and between the related intrahepatic markers CXCL10, CXCR3 and IFN-γ and liver and HIV in the liver (right hand panel). P-value < 0.05 if absolute value of Spearman’s correlation is at least 0.30, < 0.01 if at least 0.44 (n = 39). HIV human immunodeficiency virus, HBV hepatitis B virus, CA cell associated, sAg surface antigen, ALP alkaline phosphatase; GGT γ-glutamyl transferase, ALT Alanine transaminase, AST Aspartate transaminase, HMGB1 high mobility group box-1, CCL-2 C-C motif chemokine 2, sCD14 soluble CD14, LPS lipopolysaccharide, CXCL10 C-X-C motif chemokine 10, CXCR3 C-X-C motif chemokine receptor 3, IFN interferon, MPO myeloperoxidase; cccDNA covalently closed circular DNA, rcDNA relaxed circular DNA; Geq genome equivalent, TE transient elastography.

Article Snippet: Briefly, slides were incubated with antibody to CXCL10 (Rabbit; 4:100, OriGene, Rockville, Maryland), LPS (Mouse; 1:100, Abcam, Cambridge UK) or myeloperoxidase (MPO) (Rabbit; 1:1000, DAKO (Agilent) Santa Clara, CA) then rabbit or mouse Polink- 2 horseradish peroxidase (HRP) and developed with Immpact DAB (3,3′-diaminobenzidine; Vector Laboratories, Burlingame, CA), or red-AP (Vector Laboratories) then counterstained and mounted in Permount (Thermo Fisher, Waltham, MA), and scanned at high magnification (x200) using the ScanScope CS System (Aperio Technologies, Vista, CA), yielding high-resolution data from the entire tissue section.

Techniques: Virus

Journal: PLoS Pathogens

Article Title: Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection

doi: 10.1371/journal.ppat.1008744

Figure Lengend Snippet: Intrahepatic and circulating parameters associated with fibrosis outcomes by regression analysis (fibrosis in kPa by TE) and odds ratio (fibrosis by Metavir on biopsy), controlled by CD4+ T cell count.

Article Snippet: Briefly, slides were incubated with antibody to CXCL10 (Rabbit; 4:100, OriGene, Rockville, Maryland), LPS (Mouse; 1:100, Abcam, Cambridge UK) or myeloperoxidase (MPO) (Rabbit; 1:1000, DAKO (Agilent) Santa Clara, CA) then rabbit or mouse Polink- 2 horseradish peroxidase (HRP) and developed with Immpact DAB (3,3′-diaminobenzidine; Vector Laboratories, Burlingame, CA), or red-AP (Vector Laboratories) then counterstained and mounted in Permount (Thermo Fisher, Waltham, MA), and scanned at high magnification (x200) using the ScanScope CS System (Aperio Technologies, Vista, CA), yielding high-resolution data from the entire tissue section.

Techniques:

Journal: PLoS Pathogens

Article Title: Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection

doi: 10.1371/journal.ppat.1008744

Figure Lengend Snippet: (A) Liver fibrosis was significantly correlated with intrahepatic HIV DNA and with mRNA for CXCL10 and CXCR3. Plots showing correlations between liver fibrosis by TE (kPa) with intrahepatic HIV DNA, CXCL10, CXCR3 (left side panels) and between liver CXCL10 levels with intrahepatic HIV DNA, CXCR3 and peripheral AST (right side panels). The lower limit of detection for HIV RNA and HIV DNA was one copy per well. If there was no HIV PCR signal, this was recorded as zero and if there was a detectable signal but <1, this was recorded as 0.5 copies. All data points were included in the Spearman correlation. r = Spearman rank correlation coefficient. HIV human immunodeficiency virus, CXCL10 C-X-C motif chemokine 10, CXCR3 C-X-C motif chemokine receptor 3, TE transient elastography, AST Aspartate transaminase. (B) Liver fibrosis (kPa) and AST were higher in participants with detectable HIV DNA in the liver. Each symbol represents values from each participant. The lines represent the median and IQR. Comparisons were made using Wilcoxon Rank Sum test (* p<0.05).

Article Snippet: Briefly, slides were incubated with antibody to CXCL10 (Rabbit; 4:100, OriGene, Rockville, Maryland), LPS (Mouse; 1:100, Abcam, Cambridge UK) or myeloperoxidase (MPO) (Rabbit; 1:1000, DAKO (Agilent) Santa Clara, CA) then rabbit or mouse Polink- 2 horseradish peroxidase (HRP) and developed with Immpact DAB (3,3′-diaminobenzidine; Vector Laboratories, Burlingame, CA), or red-AP (Vector Laboratories) then counterstained and mounted in Permount (Thermo Fisher, Waltham, MA), and scanned at high magnification (x200) using the ScanScope CS System (Aperio Technologies, Vista, CA), yielding high-resolution data from the entire tissue section.

Techniques: Virus

Journal: PLoS Pathogens

Article Title: Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection

doi: 10.1371/journal.ppat.1008744

Figure Lengend Snippet: (A) Biopsies from people living with HIV-HBV coinfection and controls who were HIV and HBV negative were examined using immunohistochemistry (IHC) for CXCL10 (red-AP). The percentage area positive for CXCL10 staining (dark pink stain) was quantified using Photoshop CS5 and Fovea tools. Comparisons were made using the Wilcoxon Rank Sum test (* p<0.05). (i) The median and IQR for percentage area positive for CXCL10 is shown as well as (ii) representative pictures. CXCL10 staining was found within inflammatory Infiltrates, close to portal regions or blood vessels. (B) Fluorescence microscopy was performed using an RNAscope approach with a probe targeting CXCL10 (red), a nuclear stain DAPI (blue or grey) and antibodies to either (i) Myeloid cells (CD68+CD163), (ii) myeloperoxidase (MPO) or (iii) Hepatocyte (green). This demonstrates CXCL10 is predominantly located in hepatocytes. Scale bars: 100𝜇m.

Article Snippet: Briefly, slides were incubated with antibody to CXCL10 (Rabbit; 4:100, OriGene, Rockville, Maryland), LPS (Mouse; 1:100, Abcam, Cambridge UK) or myeloperoxidase (MPO) (Rabbit; 1:1000, DAKO (Agilent) Santa Clara, CA) then rabbit or mouse Polink- 2 horseradish peroxidase (HRP) and developed with Immpact DAB (3,3′-diaminobenzidine; Vector Laboratories, Burlingame, CA), or red-AP (Vector Laboratories) then counterstained and mounted in Permount (Thermo Fisher, Waltham, MA), and scanned at high magnification (x200) using the ScanScope CS System (Aperio Technologies, Vista, CA), yielding high-resolution data from the entire tissue section.

Techniques: Immunohistochemistry, Staining, Fluorescence, Microscopy, RNAscope

Journal: PLoS Pathogens

Article Title: Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection

doi: 10.1371/journal.ppat.1008744

Figure Lengend Snippet: (A) A representative flow cytometry plot showing eGFP expression (indicating infection with VSV-G-pseudotyped NL4.3Δenv eGFP HIV (MOI 0.5)) in live cells after gating on singlets and live cells, in uninfected hepatocytes (left plot) and hepatocytes infected with VSV-G-pseudotyped NL4.3Δenv eGFP HIV (MOI 0.5). (B) CXCL10 production measured by ELISA following stimulation of HepG2 (open symbols), and HBV-producing AD38 (filled symbols) hepatocyte cell lines with 500ng/ml IFN-γ plus1000ng/ml P3CSK4 and infected with VSV G-pseudotyped NL4.3Δenv eGFP HIV (MOI 0.5). HIV infection is indicated by symbols having a red border. Individual symbols represent the mean of replicates from a single experiment. The median+/-SEM for each stimulus from multiple experiments is shown. Comparisons between conditions were made using Wilcoxon Rank Sum test (* p<0.05). VSV-G-pseudotyped NL4.3Δenv eGFP HIV Vesicular stomatitis virus (VSV) glycoprotein G-pseudotyped NL4.3 virus with an envelope deletion expressing green fluorescent protein, MOI multiplicity of infection, HIV human immunodeficiency virus, CXCL10 C-X-C motif chemokine 10, IFN interferon, P3CSK4 Pam3CysSerLys4.

Article Snippet: Briefly, slides were incubated with antibody to CXCL10 (Rabbit; 4:100, OriGene, Rockville, Maryland), LPS (Mouse; 1:100, Abcam, Cambridge UK) or myeloperoxidase (MPO) (Rabbit; 1:1000, DAKO (Agilent) Santa Clara, CA) then rabbit or mouse Polink- 2 horseradish peroxidase (HRP) and developed with Immpact DAB (3,3′-diaminobenzidine; Vector Laboratories, Burlingame, CA), or red-AP (Vector Laboratories) then counterstained and mounted in Permount (Thermo Fisher, Waltham, MA), and scanned at high magnification (x200) using the ScanScope CS System (Aperio Technologies, Vista, CA), yielding high-resolution data from the entire tissue section.

Techniques: Flow Cytometry, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Virus

Journal: PLoS Pathogens

Article Title: Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection

doi: 10.1371/journal.ppat.1008744

Figure Lengend Snippet: CXCL10 production was measured by ELISA (absolute and fold change, middle and lower panel) and eGFP expression measured by flow cytometry (upper panel) following stimulation of HepG2 (open symbols), and HBV-producing AD38 (filled symbols) cells, with 500ng/ml IFN-γ plus1000ng/ml P3CSK4 and infected with VSV-G-pseudotyped NL4.3Δenv eGFP HIV. The left sided panels show eGFP expression and CXCL10 production after cells were stimulated with 500ng/ml IFN-γ plus1000ng/ml P3CSK4 and infected with VSV-G-pseudotyped NL4.3Δenv eGFP HIV with a MOI of 0.0625, 0.125, 0.25 and 0.5. Increasing MOI is indicated by the triangle at the bottom of the Fig. The right sided panels show these same parameters, following infection with VSV-G-pseudotyped NL4.3Δenv eGFP HIV (red border) with MOI of 0.5 in the presence and absence of the antiretroviral agents efavirenz (EFV), raltegravir (RAL) and the fusion inhibitor T20. Individual symbols represent the mean of replicates from a single experiment. The median+/-SEM for each stimulus from multiple experiments is shown. Comparisons between conditions were made using Wilcoxon Rank Sum test (* p<0.05). HIV human immunodeficiency virus, CXCL10 C-X-C motif chemokine 10, GFP green fluorescent protein, IFN interferon, VSV-G-pseudotyped NL4.3Δenv eGFP HIV Vesicular stomatitis virus (VSV) glycoprotein G-pseudotyped NL4.3 virus with an envelope deletion expressing green fluorescent protein, MOI multiplicity of infection, P3CSK4 Pam3CysSerLys4, efavirenz (EFV), raltegravir (RAL).

Article Snippet: Briefly, slides were incubated with antibody to CXCL10 (Rabbit; 4:100, OriGene, Rockville, Maryland), LPS (Mouse; 1:100, Abcam, Cambridge UK) or myeloperoxidase (MPO) (Rabbit; 1:1000, DAKO (Agilent) Santa Clara, CA) then rabbit or mouse Polink- 2 horseradish peroxidase (HRP) and developed with Immpact DAB (3,3′-diaminobenzidine; Vector Laboratories, Burlingame, CA), or red-AP (Vector Laboratories) then counterstained and mounted in Permount (Thermo Fisher, Waltham, MA), and scanned at high magnification (x200) using the ScanScope CS System (Aperio Technologies, Vista, CA), yielding high-resolution data from the entire tissue section.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Infection, Virus

Journal: PLoS Pathogens

Article Title: Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection

doi: 10.1371/journal.ppat.1008744

Figure Lengend Snippet: Factors driving liver disease pathogenesis in HIV-HBV co-infection In HIV-HBV co-infection we propose a model where liver fibrosis is driven by an increase in production of CXCL10 by hepatocytes (left hand side of diagram) and products of microbial translocation (right hand side of diagram). Hepatocytes produce CXCL10 and production is enhanced following infection with HBV and HIV in the presence of IFN-γ. CXCL10 recruits activated T-cells and NK cells that express CXCR3 to the liver. In the absence of antiretroviral therapy, this is an ideal environment for HIV replication which produces IFN-γ and drives further CXCL10 production. In addition, altered GI tract permeability in the setting of HIV infection leads to an increase in circulating microbial products, including LPS which binds to TLR-4 and P3S4K4 which binds to TLR-2 expressed on hepatocytes, Kupffer cells and hepatic stellate cells. This cycle of CXCL10-mediated inflammation, together with elevated LPS directly activates hepatic stellate cells (HSC) driving fibrosis. HIV human immunodeficiency virus, HBV hepatitis B, CXCL10 C-X-C motif chemokine 10, IFN interferon, NK natural killer, CXCR3 C-X-C motif chemokine receptor 3, GI gastrointestinal tract, LPS lipopolysaccharide, TLR toll like receptor, HSC hepatic stellate cells.

Article Snippet: Briefly, slides were incubated with antibody to CXCL10 (Rabbit; 4:100, OriGene, Rockville, Maryland), LPS (Mouse; 1:100, Abcam, Cambridge UK) or myeloperoxidase (MPO) (Rabbit; 1:1000, DAKO (Agilent) Santa Clara, CA) then rabbit or mouse Polink- 2 horseradish peroxidase (HRP) and developed with Immpact DAB (3,3′-diaminobenzidine; Vector Laboratories, Burlingame, CA), or red-AP (Vector Laboratories) then counterstained and mounted in Permount (Thermo Fisher, Waltham, MA), and scanned at high magnification (x200) using the ScanScope CS System (Aperio Technologies, Vista, CA), yielding high-resolution data from the entire tissue section.

Techniques: Infection, Translocation Assay, Permeability, Virus

Journal: Stroke

Article Title: Anoxia-Induced NF-kB-Dependent Upregulation of NCX1 Contributes to Ca 2+ Refilling Into Endoplasmic Reticulum in Cortical Neurons

doi: 10.1161/strokeaha.108.531962

Figure Lengend Snippet: Figure 1. Effect of OGD and OGD/reoxygenation on NCX1 transcript and protein expression and of the NF-kB inhibitor SN-50 on NCX1 protein upregulation in cortical neurons. A–D, Effect of 3 hours OGD and 3 hours OGD plus 24 hours reoxygenation on mRNA or pro- tein expression of NCX1 and their densitometric analysis. E–F, Effect of 3 hours OGD, 3 hours OGD plus SN-50 (5 mol/L) or SN-50M (5 mol/L), both preincubated for 1 hour, on NCX1 protein expression and their densitometric analysis. All the data are expressed as meansSEM (n5) and normalized on the basis of Hypoxanthine–guanine–phosphoribosyl-transferase or -actin levels. *P0.05 ver- sus normoxic cortical neurons; **P0.05 versus 3 hours OGD.

Article Snippet: Membranes were blocked with 5% nonfat dry milk in 0.1% Tween20 (2 mmol/L Tris-HCl, 50mmol/L NaCl, pH 7.5) for 2 hours at room temperature and subsequently incubated overnight at 4°C in the blocking buffer with 1:1000 antibody for NCX1 (polyclonal rabbit antibody; Swant), 1:1000 antibody for NCX2 (polyclonal rabbit antibody; Alpha Diagnostic), 1:1500 antibody for NCX3 (polyclonal rabbit antibody; Philipson’s Laboratory), 1:1000 antibody for caspase-12 (polyclonal rabbit antibody; Santa Cruz Biotechnology), 1:1000 antibody for NF-kB p65 (polyclonal rabbit antibody; Santa Cruz Biotechnology), and 1:1000 antibody for -actin (monoclonal mouse antibody; Sigma).

Techniques: Expressing

Journal: Stroke

Article Title: Anoxia-Induced NF-kB-Dependent Upregulation of NCX1 Contributes to Ca 2+ Refilling Into Endoplasmic Reticulum in Cortical Neurons

doi: 10.1161/strokeaha.108.531962

Figure Lengend Snippet: Figure 2. Effect of si-RNA against the NF-kB subunit p65 on OGD-induced NCX1 protein upregulation in cortical neurons. A–B, West- ern blots of p65 translocation from cytoplasm into nucleus after 30 minutes of OGD and 30 minutes/24 hours reoxygenation and the relative densitometric analysis, respectively. C–D, Western blots of the effect of si-RNA-p65 (20 nM) and its mismatch on p65 and -actin expression, respectively. E–F, Effect of si-RNA-p65 (20 nM) and its mismatch on NCX1 protein expression after 3 hours OGD exposure and the relative -actin expression, respectively. All the data are expressed as meansSEM (n3) and normalized on the basis the respective -actin levels. *P0.05 versus control group.

Article Snippet: Membranes were blocked with 5% nonfat dry milk in 0.1% Tween20 (2 mmol/L Tris-HCl, 50mmol/L NaCl, pH 7.5) for 2 hours at room temperature and subsequently incubated overnight at 4°C in the blocking buffer with 1:1000 antibody for NCX1 (polyclonal rabbit antibody; Swant), 1:1000 antibody for NCX2 (polyclonal rabbit antibody; Alpha Diagnostic), 1:1500 antibody for NCX3 (polyclonal rabbit antibody; Philipson’s Laboratory), 1:1000 antibody for caspase-12 (polyclonal rabbit antibody; Santa Cruz Biotechnology), 1:1000 antibody for NF-kB p65 (polyclonal rabbit antibody; Santa Cruz Biotechnology), and 1:1000 antibody for -actin (monoclonal mouse antibody; Sigma).

Techniques: Translocation Assay, Western Blot, Expressing, Control

Journal: iScience

Article Title: Monocarboxylate transporter 4 facilitates Mycobacterium tuberculosis survival through NF-κB p65-mediated interleukin-10 production.

doi: 10.1016/j.isci.2024.110238

Figure Lengend Snippet: Figure 6. Mct4 activates p65-IL-10 axis to promote Mtb survival by translocating intracellular lactate out of macrophages (A) Intracellular and extracellular lactate level was detected in Slc16a3+/+ and Slc16a3/ BMDMs at 0, 24, and 48 h of Mtb infection at MOI of 1 by lactate assay kit. (B) Intracellular and extracellular lactate level was detected in Raw 264.7 cells overexpressing Mct4 at 0, 24, and 48 h of Mtb infection at MOI of 1 by lactate assay kit. (C) Phosphorylation level of NF-kB p65 was detected by western blot in Raw 264.7 cells overexpressing Mct4 pretreated with 25 mM lactate for 3 h upon Mtb infection at MOI of 5 for 60 min. The ratio of expression of phosphorylation of NF-kB p65 and NF-kB p65 is shown in graph. b-Actin served as an internal control. (D) Intracellular mRNA and production of IL-10 were detected in Raw 264.7 cells overexpressing Mct4 pretreated with 25 mM lactate for 3 h upon Mtb infection at MOI of 1 for 48 h by qRT-PCR and ELISA analysis. b-Actin served as an internal control. (E) The intracellular Mtb load was detected in Raw 264.7 cells overexpressing Mct4 pretreated with 25 mM lactate for 3 h upon Mtb infection at MOI of 5 for 48 h by CFU assay. (A–E) Western blot results were representative of three independent experiments with similar results. Data shown were the mean G SD and are from at least three independent experiments. Data were analyzed by unpaired t test and two-way ANOVA, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Article Snippet: eFluor 450-CD80 (16-10A1) Invitrogen Cat #48-0801-82 Super Bright 600-CD86 (GL6) eBioscience Cat #63-0862-82 PE-MHC II (M5/114.15.2) TONBO Cat #50-5321-U100 IL1b Rabbit pAb ABclonal Cat #A16288 NF-kB p65 (D14E12) Rabbit mAb Cell Signaling Technology Cat #8242S p38 MAPK (D13E1) Rabbit mAb Cell Signaling Technology Cat #8690S Phospho-p38 MAPK (Thr180/Tyr182) Rabbit mAb Cell Signaling Technology Cat #4511S Stat3 (D3Z2G) Rabbit mAb Cell Signaling Technology Cat #12640 Phospho-Stat3 (Tyr705) (D3A7) Rabbit mAb Cell Signaling Technology Cat #9145 Stat1 (D1K9Y) Rabbit mAb Cell Signaling Technology Cat #14994 Phospho-Stat1 (Ser727) (D3B7) Rabbit mAb Cell Signaling Technology Cat #8826S Erk1/2 Rabbit mAb Cell Signaling Technology Cat #4695S Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) Rabbit mAb Cell Signaling Technology Cat #4370S Phospho-NF-kB p65 (Ser536) (93H1) Rabbit mAb Cell Signaling Technology Cat #3033S F4/80 Monoclonal Antibody (BM8) eBioscience Cat #14-4801-82 Phospho-NF-kB p65 (Ser536) Antibody Affinity Cat #AF2006 IL-10 neutralizing antibody eBioscience Cat #16-7102-81 Bacterial and virus strains Mtb strain of H37Rv American Type Culture Collection NO.7294 Chemicals, peptides, and recombinant proteins SB203580 Selleckchem Cat #S1076 JSH23 Selleckchem Cat #S7351 Lactate Selleckchem Cat #E4473 Experimental models: cell lines Raw 264.7 American Type Culture Collection, USA N/A

Techniques: Infection, Lactate Assay, Phospho-proteomics, Western Blot, Expressing, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Colony-forming Unit Assay

Journal: iScience

Article Title: Monocarboxylate transporter 4 facilitates Mycobacterium tuberculosis survival through NF-κB p65-mediated interleukin-10 production.

doi: 10.1016/j.isci.2024.110238

Figure Lengend Snippet: Figure 7. Potential mechanism for Mct4 to promote Mtb survival in macrophages Mtb infection increases Mct4 expression in macrophages, and Mct4 transports lactate outside of macrophages to decrease intracellular lactate, which promotes NF-kB p65 activation and IL-10 production, eventually facilitating Mtb survival.

Article Snippet: eFluor 450-CD80 (16-10A1) Invitrogen Cat #48-0801-82 Super Bright 600-CD86 (GL6) eBioscience Cat #63-0862-82 PE-MHC II (M5/114.15.2) TONBO Cat #50-5321-U100 IL1b Rabbit pAb ABclonal Cat #A16288 NF-kB p65 (D14E12) Rabbit mAb Cell Signaling Technology Cat #8242S p38 MAPK (D13E1) Rabbit mAb Cell Signaling Technology Cat #8690S Phospho-p38 MAPK (Thr180/Tyr182) Rabbit mAb Cell Signaling Technology Cat #4511S Stat3 (D3Z2G) Rabbit mAb Cell Signaling Technology Cat #12640 Phospho-Stat3 (Tyr705) (D3A7) Rabbit mAb Cell Signaling Technology Cat #9145 Stat1 (D1K9Y) Rabbit mAb Cell Signaling Technology Cat #14994 Phospho-Stat1 (Ser727) (D3B7) Rabbit mAb Cell Signaling Technology Cat #8826S Erk1/2 Rabbit mAb Cell Signaling Technology Cat #4695S Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) Rabbit mAb Cell Signaling Technology Cat #4370S Phospho-NF-kB p65 (Ser536) (93H1) Rabbit mAb Cell Signaling Technology Cat #3033S F4/80 Monoclonal Antibody (BM8) eBioscience Cat #14-4801-82 Phospho-NF-kB p65 (Ser536) Antibody Affinity Cat #AF2006 IL-10 neutralizing antibody eBioscience Cat #16-7102-81 Bacterial and virus strains Mtb strain of H37Rv American Type Culture Collection NO.7294 Chemicals, peptides, and recombinant proteins SB203580 Selleckchem Cat #S1076 JSH23 Selleckchem Cat #S7351 Lactate Selleckchem Cat #E4473 Experimental models: cell lines Raw 264.7 American Type Culture Collection, USA N/A

Techniques: Infection, Expressing, Activation Assay

Journal: PLoS Genetics

Article Title: A homozygous missense variant in CACNB4 encoding the auxiliary calcium channel beta4 subunit causes a severe neurodevelopmental disorder and impairs channel and non-channel functions

doi: 10.1371/journal.pgen.1008625

Figure Lengend Snippet: (A and B) HEK293T cells were transfected with empty vector (EV), β 1 -V5, β 4b -V5 or β 4b -L125P-V5 expression construct as indicated. Endogenous PPP2R5D and TNIK were immunoprecipitated from cell extracts using an anti-PPP2R5D (A) and an anti-TNIK antibody (B), respectively, both coupled to magnetic protein G Dynabeads. For IgG control the cell lysate from cells transfected with the β 4b -L125P-V5 mutant construct was incubated with an anti-normal rabbit IgG antibody coupled to Dynabeads. Co-precipitated β 1 -V5 and β 4b -V5 proteins were detected by immunoblotting using anti-V5-HRP and anti-β 4 antibody. A representative blot of four (A) or three (B) independent experiments each is shown.

Article Snippet: For immunoblotting and immunoprecipitation: mouse anti-CACNA2D1 (α 2 δ-1) (20A) antibody (1:500, MA3-921, Thermo Fisher Scientific), mouse anti-Ca V β 4 calcium channel antibody (1:500; 75–054, NeuroMab), mouse anti-GFP antibody (1:5000; 902601, BioLegend), anti-normal mouse IgG (1:100; 12–371, Merck Millipore) and anti-normal rabbit IgG antibody (1:100; 12–370, Merck Millipore), rabbit anti-PPP2R5D antibody (IP 1:25; WB 1:2,500; A301-098A, Bethyl Laboratories Inc.), rabbit anti-TNIK antibody (IP 1:50; WB 1:1,000; 32712, Cell Signaling Technologies), mouse anti-V5 (1:125; R960-25, Thermo Fisher Scientific), mouse anti-V5 tag horseradish peroxidase (HRP)-coupled antibody (1:5,000; R961-25, Thermo Fisher Scientific).

Techniques: Transfection, Plasmid Preparation, Expressing, Construct, Immunoprecipitation, Control, Mutagenesis, Incubation, Western Blot

Journal: Cell reports

Article Title: Unannotated microprotein EMBOW regulates the interactome and chromatin and mitotic functions of WDR5

doi: 10.1016/j.celrep.2023.113145

Figure Lengend Snippet:

Article Snippet: MYC Epitope Tag Antibody Rabbit Polyclonal , Rockland , Cat#600-401-381; RRID:AB_217927.

Techniques: Control, Recombinant, Affinity Purification, Virus, Plasmid Preparation, Quantitative Proteomics, Over Expression, Knock-Out, Software

Journal: Cell Death & Disease

Article Title: ERRα-KDM5C restrains STING enhancer activity to modulate type I interferon signaling in breast cancer progression

doi: 10.1038/s41419-026-08499-2

Figure Lengend Snippet: A MCF7 cells stably expressing Flag-HA-tagged KDM5C were subjected to affinity purification and mass spectrometry (MS) analysis. The number of peptide spectrum matches (PSMs) and unique peptides for ERRα identified to interact with KDM5C was shown as indicated. B MCF7 cells were subjected to immunoprecipitation (IP) with anti-KDM5C antibody, followed by immunoblotting (IB) analysis as indicated. C MCF7 cells were subjected to IP with anti-ERRα antibody followed by IB analysis as indicated. D MCF7 cells were subjected to immunofluorescence analysis. Nuclei (DAPI), KDM5C (green), ERRα (red). Scale bars, 10 μm. E MCF7 cells were subjected to ChIP-seq with anti-KDM5C-specific antibody. KDM5C binding sites that overlapped with ERRα were shown as indicated. F Genomic distribution of KDM5C and ERRα co-bound sites ( n = 902) is shown by pie chart. G Heat map representation of KDM5C, ERRα, H3K4me1, H3K4me2, H3K27ac, H3K9me3, and H3K27me3 ChIP-seq tag density centered on KDM5C sites that co-localized with ERRα (±3000 bp). eRNA levels on both sense (+) and antisense (−) strands on KDM5C and ERRα co-bound sites detected by GRO-seq are shown by heat map ( H ) and tag density plot ( I ) (±3000 bp). J The correlation between the ChIP-seq tag density of KDM5C and ERRα on co-bound enhancers (±1000 bp).

Article Snippet: Antibodies used are listed as following: Rabbit anti-KDM5C (Bethyl Laboratory, A301-034A, 1:100 for IP), Rabbit anti-ERRα (ABclonal, A14184, 1:100 for IP), Rabbit anti-ERRα (Cell Signaling Technology, 13826, 1:1000), Rabbit anti-KDM5C (Cell Signaling Technology, 5361, 1:1000), Mouse anti-β-ACTIN (ABclonal, AC004, 1:5000), Rabbit anti-STING (ABclonal, A3575, 1:1000), Rabbit anti-Phospho-TBK1 (Cell Signaling Technology, 5483, 1:1000), Rabbit anti-TBK1 (Cell Signaling Technology, 3504, 1:1000), Rabbit anti-Phospho-IRF3 (Cell Signaling Technology, 4947, 1:1000), Rabbit anti-Phospho-IRF3 (Cell Signaling Technology, 4302, 1:1000), Mouse anti-HA (ABclonal, AE008, 1:1000), Rabbit anti-TriMethyl-H3K4 (ABclonal, A22146, 1:100 for ChIP-seq), Rabbit anti-TriMethyl-H3K4 (ABclonal, A2357, 1:100 for ChIP and CUT&Tag), Rabbit anti-DiMethyl-H3K4 (ABclonal, A2356, for CUT&Tag), Rabbit anti-MonoMethyl-H3K4 (ABclonal, A2355, for ChIP), HRP-conjugated Goat anti-Mouse IgG (ABclonal, AS003, 1:10,000), HRP-conjugated Goat anti-Rabbit IgG (ABclonal, AS014, 1:80,000).

Techniques: Stable Transfection, Expressing, Affinity Purification, Mass Spectrometry, Immunoprecipitation, Western Blot, Immunofluorescence, ChIP-sequencing, Binding Assay

Journal: Gastroenterology

Article Title: CD151 amplifies signaling by integrin α6β1 to PI3K and induces the epithelial-mesenchymal transition in HCC cells.

doi: 10.1053/j.gastro.2011.02.008

Figure Lengend Snippet: Figure 2. High expression of CD151/integrin 6 together was associated with increased inva- sion potential of HCC cells. (A) The expression of 6, 1, and CD151 were analyzed by qRT- PCR and Western blot in 6 HCC cell lines. Immunoprecipitation showed 6 complexed with CD151. The co-localization of CD151/6 was confirmed by im- munofluorescent staining. (B) The 6 was silenced in HCCLM3 cells using specific vshRNAs. Lane 2 was validated for the most efficiency for interference of integrin 6. RT-PCR and Western blot showed the forced loss or re-expression of CD151 had little influence on 6 expres- sion. Similarity, forced loss or re- expression of 6 had little influ- ence on CD151 expression. High expression of CD151/6 together showed higher invasion potential (C) in vitro (Transwell assay) and (D) in vivo metastasis assay. Arrow refers to the meta- static loci in the lung of nude mice. Scale bar, 50 m.

Article Snippet: Primary Antibodies for Western Blot, Immunohistochemistry, and Co-immunoprecipitation Antibody Concentration for WB Concentration for IHC Concentration for IF Concentration for co-IP Specificity Company Integrin 6 1:500 1:200 1:200 4 g/mL Rat monoclonal Novus Biologicals CD151 1:150 250 1:100 1:100 25 g/mL Mouse monoclonal AbD Serotec Integrin 4 / 1:200 / / Mouse monoclonal Abcam ntegrin 1 / 1:500 / / Mouse monoclonal Abcam ntegrin 3 / 1:300 / / Mouse monoclonal Abcam -cadherin 1:5000 1:250 1:250 / Rabbit polyclonal Abcam imentin 1:100 1:500 1:500 / Rabbit polyclonal Abcam -SMA 1:500 1:500 1:500 / Rabbit polyclonal Abcam nail 1:200 1:300 1:300 / Rabbit polyclonal Abcam GF- 1R 1:50 / / / Mouse monoclonal Novus Biologicals -Met 1:200 / / / Rabbit polyclonal Abcam APDH 1:5000 / / / Mouse monoclonal Abcam AK 1:1000 / / / Rabbit polyclonal Cell Signaling Technology -FAK 1:1000 / / / Rabbit polyclonal Cell Signaling Technology KT 1:1000 / / / Rabbit polyclonal Cell Signaling Technology -AKTs473 1:1000 / 1:100 / Rabbit polyclonal Cell Signaling Technology -Akts308 1:1000 / / / Rabbit polyclonal Cell Signaling Technology ERK 1:1000 / / / Rabbit polyclonal Cell Signaling Technology p-ERK 1:1000 / / / Rabbit polyclonal Cell Signaling Technology Src 1:1000 / / / Rabbit polyclonal Cell Signaling Technology p-Src 1:1000 / / / Rabbit polyclonal Cell Signaling Technology p38 1:1000 / / / Rabbit polyclonal Cell Signaling Technology p-p38 1:1000 / / / Rabbit polyclonal Cell Signaling Technology p65 1:1000 / / / Rabbit polyclonal Cell Signaling Technology p-p65 1:1000 / / / Rabbit polyclonal Cell Signaling Technology GSK-3 1:1000 / / / Rabbit polyclonal Cell Signaling Technology p-GSK-3 1:1000 / / / Rabbit polyclonal Cell Signaling Technology PTEN 1:1000 / / / Rabbit polyclonal Cell Signaling Technology F-action / / 1:200 / Rabbit polyclonal Cell Signaling Technology Histone H3 antibody 1:5000 / / / Rabbit polyclonal Abcam Co-IP, immunoprecipitation; FAK, focal adhesion kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IF, immunofluorescence; IHC, immunohistochemistry; WB, Western blot.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Staining, Reverse Transcription Polymerase Chain Reaction, In Vitro, Transwell Assay, In Vivo

Journal: Biomolecules

Article Title: Silencing VDAC1 to Treat Mesothelioma Cancer: Tumor Reprograming and Altering Tumor Hallmarks

doi: 10.3390/biom12070895

Figure Lengend Snippet: Antibodies used in this study. Antibodies against the indicated protein, their catalogue number, source, and dilutions used in immunoblotting (WB), immunofluorescence (IF), and immunohistochemistry (IHC) experiments are presented below:

Article Snippet: Rabbit polyclonal anti-NF-kB p65 (Ser536) antibody , Bioss, MA (USA), BS-092R , - , - , 1:250.

Techniques: Western Blot, Immunofluorescence, Immunohistochemistry

Journal: Biomolecules

Article Title: Silencing VDAC1 to Treat Mesothelioma Cancer: Tumor Reprograming and Altering Tumor Hallmarks

doi: 10.3390/biom12070895

Figure Lengend Snippet: Silencing VDAC1 expression in the tumors altered inflammation. Representative immunofluorescence images of sections from si-m/hVDAC1-B-treated or si-NT-treated H226-cell-derived tumors stained and quantified for TNF-α ( A , B ), NF-kB-p65 ( C , D ), cleaved caspase-1 ( E , F ), IL-1β ( G , H ), and NRLP3 ( I , J ). Results represent the means ± SEM (n = 3) ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: Rabbit polyclonal anti-NF-kB p65 (Ser536) antibody , Bioss, MA (USA), BS-092R , - , - , 1:250.

Techniques: Expressing, Immunofluorescence, Derivative Assay, Staining

Journal: iScience

Article Title: Chimpanzee adenovirus-mediated multiple gene therapy for age-related macular degeneration

doi: 10.1016/j.isci.2023.107939

Figure Lengend Snippet: In vivo expression of eGFP, PEDF, sFlt-1, and sCD59 following IVT administration of AdCs (A) Representative fundus images of the retina from individual mice following IVT injection of 1.5×10 9 vp and 7.5×10 9 vp AdC68-eGFP. The eGFP signal could be detected from 48 h to 35 days post-injection. The dotted circles represent the edge of mouse retina. (B–D) Assessment of PEDF, sFlt-1, and sCD59 mRNA expression in retina-choroid-sclera complexes isolated from 10 mice. In each mouse, one eye was injected with AdC68-PFC (five mice for 1.5×10 9 vp and five mice for 7.5×10 9 vp) whereas the contralateral, un-injected eye served as control (only five eyes were used for analysis). At 4 days post-injection, RNA was purified from the retina-choroid complexes and real-time qPCR was conducted. Absolute number of mRNA copies were calculated using the standard curve method. (E–H) Images of western blot and quantification of the PEDF, sFlt-1, and sCD59 protein amount expressed in retina-choroid complexes of five mice. In each mouse, one eye was injected with AdC68-PFC (7.5×10 9 vp) whereas the contralateral, un-injected eye served as control. Total protein was obtained from retina-choroid-sclera complexes isolated from AdC68-PFC-treated (7.5×10 9 vp) and un-injected eyes 7 days post-injection. Antibodies against GAPDH were used for the internal control. The relative expression of PEDF, sFlt-1, and sCD59 in the un-injected eyes was set to 1. Data are expressed as mean ± SEM, and analyzed using one-way ANOVA multiple comparisons with Tukey’s method among groups in (B) and Student’s t test (two-tailed) in (C) (∗p < 0.05, ∗∗p < 0.01). PEDF, pigment epithelium-derived factor; sFlt-1, soluble fms-like tyrosine kinase-1; sCD59, soluble forms of CD59; IVT, intravitreal.

Article Snippet: Rabbit polyclonal anti-CD59 , Sino Biological , Cat#12474-RP02.

Techniques: In Vivo, Expressing, Injection, Isolation, Control, Purification, Western Blot, Two Tailed Test, Derivative Assay

Journal: iScience

Article Title: Chimpanzee adenovirus-mediated multiple gene therapy for age-related macular degeneration

doi: 10.1016/j.isci.2023.107939

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-CD59 , Sino Biological , Cat#12474-RP02.

Techniques: Virus, Recombinant, Transfection, Protease Inhibitor, Plasmid Preparation, Gel Extraction, Software

Journal: bioRxiv

Article Title: Genome-wide CRISPR activation screen identifies JADE3 as an antiviral activator of NF-kB

doi: 10.1101/2023.09.29.560128

Figure Lengend Snippet: A. Heatmap of RNA-sequencing normalized read count for select genes involved in canonical TNF-α/NF-kB signaling pathway. Coloring is done relative to each row with higher normalized read counts in red and lower normalized read counts in blue. Each box is one biological replicate. B. Vector control or JADE3 Flag A549 cells were lysed and nuclei were fractionated. Whole cell lysis and nuclear fractions were resolved using SDS-PAGE and probed for NF-kB p65. Image was spliced at blue dashed line from a single blot. Western blot shown is representative of three experiments. C. Vector control or JADE3 Flag A549 cells were treated with 20 ng/mL of TNF-α for 15 minutes, then lysed. Whole cell lysis were resolved using SDS-PAGE and probed for phosphorylated NF-kB p65 (Serine 536). Western blot shown is representative of three experiments. D. Whole cell lysis from A549 cells expressing either vector control, JADE3 Flag or JADE3 Flag lacking the indicated PHDs were resolved using SDS-PAGE and probed for phosphorylated NF-kB p65 (Serine 536). Western blot shown is representative of three experiments. E. Whole cell lysis from A549 cells expressing either a vector control or indicated JADE family members were resolved using SDS-page and probed for phosphorylated NF-kB p65 (Serine 536). Western blot shown is representative of three experiments.

Article Snippet: Antibodies used include: Rabbit anti-JADE3 (Abcam #129495) used at 1:1000, Rabbit anti-NF-kB p65 (Cell Signaling #8242) used at 1:1000, Rabbit anti-phospho-NF-kB p65 Ser536 (Cell Signaling #3033) used at 1:1000, Rabbit anti-H3 (Cell Signaling), anti-Flag HRP (Sigma), anti-rabbit IGG HRP (Sigma), anti-GAPDH HRP (Sigma)

Techniques: RNA Sequencing, Plasmid Preparation, Control, Lysis, SDS Page, Western Blot, Expressing

Journal: Cell Reports

Article Title: Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation

doi: 10.1016/j.celrep.2021.109101

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-CEBPβ , Bethyl , Cat#A302-738A; RRID: AB_10627809.

Techniques: Recombinant, Multiplex sample analysis, Cell Isolation, Activation Assay, Staining, Flow Cytometry, Expressing, Reverse Transcription, Transfection, TA Cloning, Plasmid Preparation, Methylation, Immunoprecipitation, Purification, DNA Library Preparation, Library Quantification, Control, Sequencing, Methylation Sequencing, Amplification, Software

Journal: Journal of proteome research

Article Title: CaMKII Phosphorylation of Na V 1.5: Novel in Vitro Sites Identified by Mass Spectrometry and Reduced S516 Phosphorylation in Human Heart Failure

doi: 10.1021/acs.jproteome.5b00107

Figure Lengend Snippet: Purification of NaV1.5 for LC–MS/MS analysis. (A) Biochemical fractionations of NaV1.5 expressing HEK293 cells (four fractions from n = 2 representative samples: Cyto (two consecutive cytoplasmic washes), Mb (membrane), and Urea (detergent insoluble). An equal amount of a nonfractionated whole-cell lysate (RIPA) is provided for comparison. Membranes were probed for NaV1.5 and GAPDH. (B) Proteomics workflow for identification and label-free quantification of in vitro CaMKII phosphorylation sites on human NaV1.5. (C) NaV1.5 immunoprecipitated (IP) from crude Mb (membrane) fractionations with either NaV1.5 or control IgG antibody, reacted with or without CaMKII, and probed with pS516 or total NaV1.5 antibody. Starting lysates and flow-throughs (FT) are shown for comparison. (D) Representative full 1-D SDS-PAGE gel of immunopurified NaV1.5. NaV1.5 gel bands running at ~220 kDa (boxed in red) were cut out for LC–MS/MS. MW = molecular weight marker.

Article Snippet: For Western blot analysis of IPs, Na V 1.5 IPs and in vitro phosphorylation reactions were performed as previously described with the addition of an IgG control IP (rabbit IgG antibody, Santa Cruz no. sc-2027).

Techniques: Purification, Liquid Chromatography with Mass Spectroscopy, Expressing, In Vitro, Immunoprecipitation, SDS Page, Molecular Weight, Marker