MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot

Antibody (Ab) validation is the procedure in which an Ab is thoroughly assayed for sensitivity and specificity in a given application. Validation of Abs against post-translationally modified (PTM) targets is particularly challenging because it requires specifically prepared antigen. Here we describe a novel validation method using surrogate proteins in a Western blot. The surrogate protein, which we termed ‘MILKSHAKE,’ is a modified maltose binding protein enzymatically conjugated to a peptide from the chosen target that is either modified or nonmodified at the residue of interest. The certainty of the residue’s modification status can be used to confirm Ab specificity. This method also allows for Ab validation even in the absence or limited availability of treated cell lysates.

Long Noncoding RNA SNHG4 Remits Lipopolysaccharide‐Engendered Inflammatory Lung Damage by Inhibiting METTL3 - Mediated m6A Level of STAT2 mRNA

Background: Emerging evidence suggests that long non coding RNA (lncRNA) small nucleolar RNA host gene 4 (SNHG4) has become a new insight into lipopolysaccharide (LPS) - induced microglia inflammation, its role in neonatal pneumonia (NP) remains to be largely unrevealed.Methods: RT-qPCR was used to determine SNHG4 and METTL3 expression in the serum from NP patients and normal volunteers, as well as in WI-38 cells treated with LPS. The SNHG4 overexpression vector (pcDNA-SNHG4) was transfected into LPS - treated cells. CCK-8, Transwell, annexin V-FITC/PI and ELISA assays were used to determine cell proliferation, migration, apoptosis and contents of IL-6, TNF-α, SOD and MDA, respectively. The level of SNHG4 in the promoter region of METTL3 was assessed with RIP assay. m6A quantitative analysis illustrated the m6A level with or without SNHG4 overexpression or METTL3 silencing. Bioinformatics analysis and RIP-PCR were used to predict and validate YTHDF1 - mediated m6A levels on signal transducer and activator of transcription 2 (STAT2) mRNA in METTL3 inhibited cells. Then rescue experiments were performed to explore effects of SNHG4 and METTL3 or STAT2 on LPS-treated cell functions. Subsequently, in vivo functional experiments were performed to investigate the role of SNHG4 in LPS induced pneumonia in mice. Results: SNHG4 was downregulated and METTL3 was upregulated in NP patients and LPS-treated cells. SNHG4 overexpression facilitated cell proliferation, migration and SOD concentration, and inhibited apoptosis and IL-6, TNF-α and MDA contents. Mechanistically, SNHG4 bound with METTL3 and downregulated METTL3 expression. Besides, total m6A modification level was lower in the SNHG4 overexpressed or METTL3 inhibited cells. METTL3 interference reduced m6A levels of STAT2 mRNA, decreased STAT2 mRNA stability and promoted STAT2 translation level. METTL3 or STAT2 upregulated reversed the effects of SNHG4 overexpression on LPS - treated cell functions. Conclusions: This study reveals that SNHG4 promotes LPS induced inflammation in human lung fibroblasts and mouse lung tissues in vitro and in vivo by inhibiting METTL3 - mediated m6A level of STAT2 mRNA, which may provide a potential therapeutic mechanism for NP.

Synthesis and Antiproliferative Activity of Chlorinated Maprotiline Analogues

A novel chlorinated tetracyclic compound 13 of the class ethanoanthracene, as analogue of maprotiline, was prepared via multistep syntheses. The tetracyclic key intermediates 4 and 5 with its [2.2.2] system were built via a Diels-Alder reaction between acrolein and 1,8-dichloroanthracene.The synthesized chlorinated maprotiline analogues 6 , 7 and 13 as well intermediates 4 and 5 exerted antiproliferative activity against cancer cell lines A549 and HepG2 at low micromolar concentrations. In addition, the intermediates 4 and 5 exerted high antiproliferative activity against HCT cell line. Interestingly, the intermediate 4 was the most active against all treated cell lines.

Synthesis and Antiproliferative Activity of Chlorinated Maprotiline Analogues

A novel chlorinated tetracyclic compound 13 of the class ethanoanthracene, as analogue of maprotiline, was prepared via multistep syntheses. The tetracyclic key intermediates 4 and 5 with its [2.2.2] system were built via a Diels-Alder reaction between acrolein and 1,8-dichloroanthracene.The synthesized chlorinated maprotiline analogues 6, 7 and 13 as well intermediates 4 and 5 exerted antiproliferative activity against cancer cell lines A549 and HepG2 at low micromolar concentrations. In addition, the intermediates 4 and 5 were also exerted high antiproliferative activity against HCT cell line. Interestingly, the intermediate 4 was the most active against all treated cell lines.