ccl1 protein  (MedChemExpress)

Journal: Cell proliferation

Article Title: Inhibition of Alkbh5 Attenuates Lipopolysaccharide-Induced Lung Injury by Promoting Ccl1 m6A and Treg Recruitment.

doi: 10.1111/cpr.70032

Figure Lengend Snippet: FIGURE 4 | Alkbh5 directly binds to Ccl1 mRNA. A, RNA-seq analysis to identify the top 10 differentially expressed genes in Alkbh5 KO mouse lung tissues and Alkbh5-silenced MLE12 cells; (B, C) qPCR and WB to detect the mRNA and protein levels of Ccl1 in mouse lung tissues and MLE12 cells; (D) ELISA to detect the levels of Ccl1 in mouse serum; (E) m6A modification of Ccl1 mRNA was detected by MeRIP-qPCR analysis using anti- IgG and anti-m6A antibodies; (F) relative enrichment of Ccl1 mRNA associated with Alkbh5 protein was identified by RIP assays using anti-IgG and anti-FLAG antibodies; (G) immunoblotting of Alkbh5 in IgG and Flag-Ccl1 mRNA groups; (H) Cy3-labelled Ccl1 mRNA FISH and anti-Alkbh5 co-localization in lung tissues and cells. Each group contained six mice, and cell experiments were repeated at least three times. Data are presented as dot and violin plots and were statistically analysed using one-way (B–E) or two-way (E) ANOVA followed by Tukey's multiple comparison, or an- alysed by the unpaired t-test (E). *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: For pharmacological interventions, mice received intraperitoneal injections of the Alkbh5 antagonist DDO- 2728 (5 mg/kg, MCE) or recombinant mouse Ccl1 protein (mCcl1) (10 μg/kg, R&D system) 24 h prior to LPS injection.

Techniques: RNA Sequencing, Enzyme-linked Immunosorbent Assay, Modification, Western Blot, Comparison

Journal: Cell proliferation

Article Title: Inhibition of Alkbh5 Attenuates Lipopolysaccharide-Induced Lung Injury by Promoting Ccl1 m6A and Treg Recruitment.

doi: 10.1111/cpr.70032

Figure Lengend Snippet: FIGURE 5 | Alkbh5 deficiency enhances Ccl1 mRNA stability via an m6A-dependent manner. (A) SRAMP website analysis of m6A modifica- tion sites on Ccl1 mRNA; (B) schematic diagram of pGL3 luciferase reporter vectors containing 5′-UTR, CDS and 3′-UTR sequences; (C) the Alkbh5 overexpression vector was co-transfected with the constructed pGL3 luciferase reporter vector into 293T cells to analyse luciferase activity; (D) four mutated pGL3 luciferase reporter vectors were constructed; (E) the Alkbh5 overexpression vector was co-transfected with the constructed pGL3 luciferase reporter vector into 293T cells to analyse luciferase activity; (F) qPCR to detect the half-life of Ccl1 mRNA in cells. Cell experiments were repeated at least three times. Data are presented as dot and violin plots and were statistically analysed using two-way ANOVA followed by Tukey's multiple comparison (C–F). ***p < 0.001, ****p < 0.0001.

Article Snippet: For pharmacological interventions, mice received intraperitoneal injections of the Alkbh5 antagonist DDO- 2728 (5 mg/kg, MCE) or recombinant mouse Ccl1 protein (mCcl1) (10 μg/kg, R&D system) 24 h prior to LPS injection.

Techniques: Luciferase, Over Expression, Plasmid Preparation, Transfection, Construct, Activity Assay, Comparison

Journal: Cell proliferation

Article Title: Inhibition of Alkbh5 Attenuates Lipopolysaccharide-Induced Lung Injury by Promoting Ccl1 m6A and Treg Recruitment.

doi: 10.1111/cpr.70032

Figure Lengend Snippet: FIGURE 6 | Alkbh5 deficiency increased the Ccl1-mediated recruitment of Tregs. (A) Flow cytometry was used to detect the number of CD4+Foxp3+ Tregs in lung tissues; (B) Immunofluorescence to detect the number of Foxp3-positive cells, a Treg marker, in mouse lung tissues; (C) ELISA to detect the levels of Treg cytokines Tgfb1 and Il4 in mouse serum; (D) Ccl1-specific antagonist R243 (0.3 mg/kg) was used to treat KO mice; (E) ELISA to detect the levels of KL-6, SP-D, TNF-alpha and CRP in mouse serum; (F–I) HE staining (F), PAS staining (G), Masson's trichrome stain- ing (H) and TUNEL (I) assays to detect pathological changes, immune cell infiltration, fibrosis levels and apoptosis in lung tissues; (J) flow cytometry to detect the number of CD4+Foxp3+ Tregs in lung tissues; (K) immunofluorescence to detect the number of Foxp3-positive cells, a Treg marker, in mouse lung tissues. Each group contained six mice. Data are presented as dot and violin plots and were statistically analysed using one-way ANOVA followed by Tukey's multiple comparison (A–C), or analysed by the unpaired t-test (E–K). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: For pharmacological interventions, mice received intraperitoneal injections of the Alkbh5 antagonist DDO- 2728 (5 mg/kg, MCE) or recombinant mouse Ccl1 protein (mCcl1) (10 μg/kg, R&D system) 24 h prior to LPS injection.

Techniques: Flow Cytometry, Immunofluorescence, Marker, Enzyme-linked Immunosorbent Assay, Staining, TUNEL Assay, Comparison