Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Enhanced large conductance K + channel activity contributes to the impaired myogenic response in the cerebral vasculature of Fawn Hooded Hypertensive rats

doi: 10.1152/ajpheart.00636.2013

Figure Lengend Snippet: Comparison of BK-α- and BK-β-subunit expression in cerebral vessel homogenates isolated from FHH and FHH.1BN rats. BK-α- and BK-β-subunits were probed on different blots. The same blots were then reprobed for β-actin. A: representative bands corresponding to the molecular weight of the BK-α-subunit (∼100 kDa), BK-β-subunit (∼25 kDa), and β-actin (∼42 kDa) in cerebral vessels obtained from FHH and FHH.1BN rats (n = 8 animals each; 3 sets of experiments). B: a comparison of the expression of α- and β-subunits. Numbers in parentheses indicate the number of samples studied.

Article Snippet: After transfer, the membrane was blocked with TBS-T buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween, and a 5% blocking powder (Bio-Rad) at 4°C for 1 h. The blot was probed with primary antibody against BK α- and β-subunit [1:500 and 1:200, respectively; polyclonal rabbit Anti-K Ca 1.1, to amino acids 1184–1200 and Anti-sloβ1 (KCNMB1); Alomone Labs, Jerusalem, Israel] overnight at 4°C.

Techniques: Expressing, Isolation, Molecular Weight

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

Article Title: Role of IL-6-IL-27 Complex in Host Antiviral Immune Response.

doi: 10.4049/jimmunol.2100179

Figure Lengend Snippet: FIGURE 1. Interactions between the subunits of IL-6 and IL-27 in vivo and in vitro. (AC) CoIP and immunoblotting of serum samples were performed with the indicated Abs. IL-6 interacted with IL-27A (A), IL-6 interacted with EBi3 (B), and IL-27A interacted with EBi3 (C). (DG) HEK293T cells were transfected with the indicated plasmids for 24 h. CoIP and immunoblotting were performed with the indicated Abs. IL-6-3HA interacted with IL-27A-V5 (D), IL-6-3HA interacted with EBi3-Myc (E), IL-27A-V5 interacted with EBi3-Myc (F), and IL-6-3HA interacted with IL-27-V5 (G). (H) Colocalization of IL-6 and IL-27 was detected by confocal microscopy. A549 cells were transfected with IL-6-eGFP-KDEL and IL-27-DsRed-KDEL for 24 h, and the nuclei were stained with DAPI before observation using confocal microscopy. All experiments were repeated at least three times with consistent results.

Article Snippet: Abs against V5 tag (66007-1-Ig), EBi3 (12371-1-AP), myxovirus resistance A (MxA; 13750-1-AP), MAVS (14341-1-AP), TGFb-activated kinase 1 (12330-2-AP), IRF3 (11312-1-AP), NF-kB p65 (10745- 1-AP), NF-kB p50 (14220-1-AP), Lamin A/C (10298-1-AP), GAPDH (60004-1-Ig), and b-actin (60008-1-Ig) were purchased from ProteinTech Group (Wuhan, China).

Techniques: In Vivo, In Vitro, Western Blot, Transfection, Confocal Microscopy, Staining

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

Article Title: Role of IL-6-IL-27 Complex in Host Antiviral Immune Response.

doi: 10.4049/jimmunol.2100179

Figure Lengend Snippet: FIGURE 2. Construction of the IL-6IL-27 complex and its antiviral activity. (A) A schematic of the IL-6IL-27 construction via flexible self-cleaved linker is shown. Immunoblotting analysis of IL-6, EBi3, IL-27A, and b-actin of HEK293T cells transfected with empty vector pcDNA3.1(1) or IL-6IL-27 complex expression plasmids for 24 h. (B) A549 cells were transfected with indicated plasmids for 24 h followed by H1N1 infection (MOI = 1) for 12 h. The NP-specific mRNA, cRNA, and vRNA levels were measured using QRT-PCR. (C) RD cells were transfected with the indicated plasmids for 24 h fol- lowed by EV71 infection (MOI = 1) for 24 h. The VP1 mRNA levels were measured using QRT-PCR. (D) Vero cells were (Figure legend continues)

Article Snippet: Abs against V5 tag (66007-1-Ig), EBi3 (12371-1-AP), myxovirus resistance A (MxA; 13750-1-AP), MAVS (14341-1-AP), TGFb-activated kinase 1 (12330-2-AP), IRF3 (11312-1-AP), NF-kB p65 (10745- 1-AP), NF-kB p50 (14220-1-AP), Lamin A/C (10298-1-AP), GAPDH (60004-1-Ig), and b-actin (60008-1-Ig) were purchased from ProteinTech Group (Wuhan, China).

Techniques: Activity Assay, Western Blot, Transfection, Plasmid Preparation, Expressing, Infection, Quantitative RT-PCR

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

Article Title: Role of IL-6-IL-27 Complex in Host Antiviral Immune Response.

doi: 10.4049/jimmunol.2100179

Figure Lengend Snippet: FIGURE 5. The IL-6IL-27 complex interacts with MAVS. (AC) A549 cells were transfected with increasing amounts of the indicated expression plas- mids (wedge; 100, 200, and 300 ng) of the IL-6IL-27 complex expression plasmid and luciferase reporter plasmids containing the IFN-b (A), NF-kB (B), and ISRE (C) promoter for 24 h followed by VSV infection (MOI = 1) for 24 h. Reporter assays were performed. pRL-TK was used as an internal control. (DI) HEK293T cells were transfected with the indicated plasmids for 24 h. CoIP and immunoblotting were performed with the indicated Abs. IL-6-3HA interacted with Flag-MAVS (D), IL-27A-V5 interacted with Flag-MAVS (E), EBi3-Myc interacted with Flag-MAVS (F), IL-6-HA interacted with Flag- TBK1 (G), IL-27A-V5 interacted with Flag-TBK1 (H), and EBi3-Myc interacted with Flag-TBK1 (I). All experiments were repeated at least three times with consistent results.

Article Snippet: Abs against V5 tag (66007-1-Ig), EBi3 (12371-1-AP), myxovirus resistance A (MxA; 13750-1-AP), MAVS (14341-1-AP), TGFb-activated kinase 1 (12330-2-AP), IRF3 (11312-1-AP), NF-kB p65 (10745- 1-AP), NF-kB p50 (14220-1-AP), Lamin A/C (10298-1-AP), GAPDH (60004-1-Ig), and b-actin (60008-1-Ig) were purchased from ProteinTech Group (Wuhan, China).

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Infection, Control, Western Blot

Journal: Journal of Hematology & Oncology

Article Title: The LINC00623/NAT10 signaling axis promotes pancreatic cancer progression by remodeling ac4C modification of mRNA

doi: 10.1186/s13045-022-01338-9

Figure Lengend Snippet: NAT10 functions as a downstream mediator of LINC00623by remodeling N4-acetylcytidine (ac4C) modification of mRNA. a Representative images of IHC staining with an anti-NAT10 antibody in PDAC, pancreatic intraepithelial neoplasia (PanIN) or matched adjacent normal pancreatic tissues (NP). b The mRNA level of LINC00623 was positively correlated with the protein level of NAT10 in PDAC tissues ( n = 93). c Kaplan‒Meier survival curve of two groups of patients with PDAC ( n = 93): NAT10 (+), patients with high NAT10 expression; NAT10 (-), patients with low NAT10 expression. The expression of NAT10 was determined by IHC staining. d Schematic diagram of the effects of NAT10 on the stability and translation efficiency of mRNA by catalyzing ac4C modification. e Metagene profile showing the distribution of NAT10 peaks across full-length transcripts containing the 5′UTR, CDS, and 3′UTR. BxPC-3 cell lysates were used for the NAT10 RIP assay. f Metagene profile showing the distribution of ac4C peaks across full-length transcripts containing the 5′UTR, CDS, and 3′UTR. BxPC-3 cell lysates were also used for acRIP. g Venn diagram showing the downstream target genes regulated by NAT10 via ac4C modification in BxPC-3 cells. Group 1: The gene set enriched in NAT10 RIP-seq (RIP-seq); Group 2: The set of target genes enriched in parental cells but not in NAT10-silenced cells according to acRIP-seq (acRIP-seq); Group 3: The genes upregulated or downregulated in NAT10-silenced cells compared with control cells (RNA-seq); Group 4: The mRNA transcripts displaying differences in translation efficiency in NAT10-silenced cells (Ribo-seq). h Functional annotation and pathway enrichment analysis of the predicted downstream target genes of NAT10 according to the Metascape database. i The relative mRNA levels of NAT10, KCNN4, LAMB3 and PHGDH were measured by RT-qPCR. j The protein levels of KCNN4, LAMB3 and PHGDH were determined by Western blotting. k RT-qPCR was used to detect the relative enrichment of KCNN4 , LAMB3 and PHGDH mRNAs in NAT10 RIP products. l RT-qPCR was used to detect the relative enrichment of KCNN4, LAMB3 and PHGDH mRNAs in acRIP products. i – l BxPC-3 cells were transfected with NAT10 silencing and control plasmids. Cell lysates were harvested for RIP assays. IgG was used as the isotype control. The values indicate the mean ± SD of three independent experiments. P values are shown as * P < 0.05; ** P < 0.01; *** P < 0.001. Independent Student’s t test

Article Snippet: The sections were then incubated with specific primary antibodies against PCNA (1:200 dilution, ab15497, Abcam, Cambridge, UK), NAT10 (1:500 dilution, ab194297, Abcam), Vimentin (1:500 dilution, ab92547, Abcam), E-cadherin (1:500 dilution, 20874-1-AP, Proteintech), N-cadherin (1:100 dilution, ab76011, Abcam), LAMB3 (1:500 dilution, 26795-1-AP, Proteintech), PHGDH (1:500 dilution, 14719-1-AP, Proteintech) and KCNN4 (1:200 dilution, 23271-1-AP, Proteintech) at 4 °C overnight.

Techniques: Modification, Immunohistochemistry, Expressing, Control, RNA Sequencing, Functional Assay, Quantitative RT-PCR, Western Blot, Transfection