Journal: Free radical biology & medicine

Article Title: Loss of Camk2n1 aggravates cardiac remodeling and malignant ventricular arrhythmia after myocardial infarction in mice via NLRP3 inflammasome activation.

doi: 10.1016/j.freeradbiomed.2021.03.014

Figure Lengend Snippet: Fig. 4. The expression of NLRP3 inflammasome was upregulated in Camk2n1¡/¡ mice 3 days post-MI compared to WT littermates. (A) GO enrichment analysis of differentially expressed genes, as identified by RNA-seq in border zone of WT and Camk2n1−/− mice 3 days post-MI and top 10 of up-regulated inflammation-related enriched GO biological process terms were listed (n = 3 for each group). (B) Heat map for upregulated inflammatory markers. (C–H) Vali dation for the RNA-seq results of NLRP3 (C), ASC (D), CCL2 (E), CXCL1 (F), TNF-α (G) and IL-6 (H) by RT-PCR (n = 5 for each group). Data in (C–H) were analyzed by one-way ANOVA with Bonferroni’s multiple comparisons test. *P < 0.05 vs sham group, **P < 0.01 vs sham group, #P < 0.05 vs WT MI group, ##P < 0.01 vs WT MI group.

Article Snippet: After being blocked by 5% filtered non-fat milk for 1 h, the bands were incubated with the primary antibodies overnight at 4 ◦C, including Total-CaMKIIδ (1:1000; ab181052; Abcam, Cambridge, UK), PhosphoCaMKIIδ (1:1000; ab182647; Abcam), NLRP3 (1:1000; ab214185; Abcam), Caspase-1 (1:1000; 22915-1-AP; Proteintech Group, Inc, Chicago, IL), ASC (1:1000; 10500-1-AP; Proteintech), Camk2n1 (1:300; SAB1302411; Sigma), IL-18 (1:1000; ab71495; Abcam), IL-1β (1:1000; ab205924; Abcam), GAPDH (1:1000; ab8245; Abcam), α-smooth muscle actin (α-SMA, 1:1000; ab7817; Abcam), collagen-I (1:1000; ab34710; Abcam), Connective Tissue Growth Factor (CTGF, 1:1000; ab6992; Abcam), Phospho-Erk1/2 (1:1000; 4377; Cell Signaling Technology), Phospho-JNK (1:1000; 9255; Cell Signaling Technology), Phospho-p38 (1:1000; 4511; Cell Signaling Technology), Erk1/2 (1:1000; 9102; Cell Signaling Technology), JNK (1:1000; 9252; Cell Signaling Technology), p38 (1:1000; 8690; Cell Signaling Technology), Ryanodine receptor 2 (RYR2, 1:1000; 19765-1-AP; Proteintech), Phospho-RYR2 (1:1000; A010-31AP; Badrilla), SERCA2A (1:1000; ab150435; Abcam), Phospholamban (1:1000; ab219626; Abcam), Phospho-Phospholamban (1:1000; AP0910; ABclonal), Bax (1:4000; 50599-2-Ig; Proteintech), Bcl-2 (1:1000; 12789-1-AP; Proteintech), Caspase-3 (1:1000; 9661s; Cell Z. Wei et al. Free Radical Biology and Medicine 167 (2021) 243–257 Signaling Technology).

Techniques: Expressing, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular medicine reports

Article Title: MEK inhibitor enhanced the antitumor effect of oxaliplatin and 5‑fluorouracil in MEK1 Q56P‑mutant colorectal cancer cells.

doi: 10.3892/mmr.2018.9730

Figure Lengend Snippet: Figure 4. Gene expression induced by U0126 combined with or without oxaliplatin/5‑FU. (A) ERCC1 mRNA levels in SW48 cells were examined by RT‑qPCR following treatment with U0126 and/or oxaliplatin. (B) TYMS mRNA levels in SW48 cells were examined by RT‑qPCR following treatment with U0126 and/or 5‑FU. The graph depicts the fold change in ERCC1/TYMS levels normalized to β‑actin levels. Data are presented as means ± standard deviation. Western blot analysis results of (C) ERCC1 and (D) TYMS protein expression levels in cells treated with U0126 in combination with oxaliplatin or 5‑FU, respectively. **P<0.01 vs. control group; ##P<0.01 vs. oxaliplatin/5‑FU‑treated group. 5‑FU, 5‑fluorouracil; ERCC1, excision repair cross‑complementation group 1; TYMS, thymidylate synthase; RT‑qPCR, reverse transcription‑quantitative polymerase chain reaction.

Article Snippet: Excision repair cross-complementation group 1 (ERCC1) rabbit polyclonal antibody (cat. no. 14586‐1‐AP) and TYMS rabbit polyclonal antibody (cat. no. 15047‐1‐AP) were purchased from ProteinTech Group, Inc. (Wuhan, China).

Techniques: Gene Expression, Standard Deviation, Western Blot, Expressing, Control, Polymerase Chain Reaction

Journal: The Journal of biological chemistry

Article Title: Cryopyrin-induced interleukin 1beta secretion in monocytic cells: enhanced activity of disease-associated mutants and requirement for ASC.

doi: 10.1074/jbc.M401178200

Figure Lengend Snippet: FIG. 4. Cryopyrin requires oligomerization for ASC binding and IL-1 secretion. A, schematic representation of the cryopyrinPD-Fpk3 or Fpk3 vector. B, 1 108 THP-1 cryopyrinPD-Fpk3 and Fpk3 stable cells were immunoprecipitated (IP) with polyclonal myc Ab (top panel), and total lysates (bottom panel) from stable cells were immunoblotted with monoclonal ASC Ab. C, 0.5 106 THP-1 cells stably expressing with cryopyrinPD-Fpk3 or Fpk3 vector in the presence () or absence () of Fpk3 ligand AP1510 for 0, 4, and 8 h followed by detection of IL-1 secretion. The results are given as the mean S.D. of triplicate cultures.

Article Snippet: The antibodies used for immunoprecipitation and immunodetection were anti-FLAG monoclonal (Santa Cruz), anti-Myc polyclonal (Santa Cruz), anti-ASC polyclonal ( ProSci), anti-ASC monoclonal (21), and anti-tubulin monoclonal (Sigma).

Techniques: Binding Assay, Plasmid Preparation, Immunoprecipitation, Stable Transfection, Expressing