Journal: Nucleic Acids Research

Article Title: Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation

doi: 10.1093/nar/gks504

Figure Lengend Snippet: Isolation of a KARRE from the upstream 3′ splice site of the exon 5a of Snap25 gene based on forskolin reduction of 5a/5b ratios in PC12 cells during neuronal differentiation. ( A ) Forskolin-induced neuronal differentiation of PC12 cells. Shown are representative images of PC12 cells without or with forskolin addition for 18 h. ( B ) Diagram of the alternative splicing of the exons 5a and 5b of the Snap25 gene. Arrows: sites of restriction enzymes NdeI (5a) and AvaII (5b) used to digest the PCR products. Arrowheads: PCR primers. ( C ) RT-PCR products from untreated (−) and treated PC12 cells. ETOH: ethanol, vehicle for forskolin and Dex (dexamethasone); H89: a PKA-specific inhibitor; NC: PCR negative control; M: molecular size marker. ( D) Diagram of the deletion/replacement mutants of Snap25 splicing reporters and their exon inclusion levels in the presence of PKAm or active PKA. Asterisks: indicating P value levels in one tail, paired Student’s t -test (*** P < 0.001, * P < 0.05, n ≥ 3). ( E) An agarose gel showing the PKA response of the heterologous S175NK reporter containing the 17 nt Snap25a element upstream of a stronger exon (175NK, about 75% inclusion with PKAm coexpression).

Article Snippet: Some cultures were pretreated with 10 µM of H89 ( N -[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamidedihydrochloride, Sigma, #B1427) for 10 min. To differentiate products of Snap25 endogenous mRNA transcripts from exons 5a and 5b, we digested 1.0 µl of 32 P-labelled-PCR products with AvaII and NdeI restriction enzymes in buffer 4 (New England Biolabs) in a 10 -µl reaction at 37°C for 1 h and run in 6% denaturing polyacrylamide gel electrophoresis (PAGE) gel.

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Negative Control, Marker, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: Activin A correlates with the worst outcomes in COVID-19 patients, and can be induced by cytokines via the IKK/NF-kappa B pathway

doi: 10.1101/2021.02.04.429815

Figure Lengend Snippet: Activin A production was measured in conditioned medium by enzyme-linked immunosorbent assay (ELISA). BTSMC (Bronchial/tracheal smooth muscle) cells, NHLF (Normal human Lung Fibroblasts) and PASMC (Pulmonary artery smooth muscle cells) were serum starved overnight and then treated with 10ng/ml IL1 α or TNF α for five days in the presence or absence of 100uM Dexamethasone, 100nM anti-Activin A antibody REGN2477 or isotype control REGN1945. Conditioned medium was diluted at 1:10 for ELISA. The experiment was repeated twice, with a consistent result.

Article Snippet: To study the effect of pro-inflammatory cytokines IL-1 α and TNF α on Activin A production and whether Dexamethasone and anti-Activin A can rescue, cells were pretreated with 100μM dexamethasone (Sigma-Aldrich Inc, #D2915), 100nM REGN1945 (isotype control) or 100nM REGN2477 (anti-Activin A) for 10 minutes, and then treated with or without 10ng/ml IL-1 α (R&D Systems, #200-LA-CF) or TNF α (R&D Systems, #210-TA-CF) for five days.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: bioRxiv

Article Title: Activin A correlates with the worst outcomes in COVID-19 patients, and can be induced by cytokines via the IKK/NF-kappa B pathway

doi: 10.1101/2021.02.04.429815

Figure Lengend Snippet: (A) Bronchial/tracheal smooth muscle cells (SMC) and (B) pulmonary artery smooth muscle cells (SMC) were treated for 24 hours with 100ng/ml of TNF α or IL1 α in combination with pharmacological inhibitors for IKK (3uM Withaferin A), p38 (0.3uM SB203580) and JNK (30uM SP600125). A combination of IKK and p38 inhibitors (same concentrations), a combination of IKK and JNK inhibitors (same concentrations) and a combination of p38 and JNK inhibitors (same concentrations) was also used. DMSO was used as a negative control for inhibitor treatments. Treatment with sterile water only (Mock) was used as an additional control. Activin A levels were quantified in conditioned media by ELISA. N=3 for each treatment. Significant Bonferroni-corrected pairwise comparisons with DMSO within each treatment condition are labeled. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To study the effect of pro-inflammatory cytokines IL-1 α and TNF α on Activin A production and whether Dexamethasone and anti-Activin A can rescue, cells were pretreated with 100μM dexamethasone (Sigma-Aldrich Inc, #D2915), 100nM REGN1945 (isotype control) or 100nM REGN2477 (anti-Activin A) for 10 minutes, and then treated with or without 10ng/ml IL-1 α (R&D Systems, #200-LA-CF) or TNF α (R&D Systems, #210-TA-CF) for five days.

Techniques: Negative Control, Sterility, Control, Enzyme-linked Immunosorbent Assay, Labeling

Journal: NPJ Vaccines

Article Title: Epitope-specific immunity against Staphylococcus aureus coproporphyrinogen III oxidase

doi: 10.1038/s41541-020-00268-2

Figure Lengend Snippet: a S. aureus CgoX structure was modulated from B. subtillis (3I6D.pdb). The linear epitope of mAb D3 (red) was identified by microarray technology using overlapping 13mer CgoX peptides (Supplementary Fig. ). Protein structure was visualised by EzMol2.1. b Alanine scan of epitope peptides for binding analysis of anti-CgoX mAb D3. Single amino acid positions of the D3 epitope were consecutively replaced by alanine (red in right panel). Immobilised peptides were stained by anti-CgoX mAb D3 detected with anti-mIgG-HRP. Data are presented as mean ± s.d. ( n = 2 technical replicates). c Allele frequencies of anti-CgoX mAb D3 epitope. Genome sequences of S. aureus clinical isolates were analysed for epitope aa sequence using the RidomSeqsphere core genome multi locus sequence typing (cgMLST) database. Amino acids interacting with paratope of anti-CgoX mAb D3 according to alanine scan are marked in red. Amino acid differences from identified epitope peptide sequence are marked in blue. Frequencies of alleles with non-restricted binding of anti-CgoX mAb D3 are marked in green. d Uniqueness of the CgoX D3 epitope in S. aureus . Sequence alignment of CgoX from S. aureus with PPOX from H. sapiens and M. musculus . CgoX D3 epitope is depicted in yellow. e Competition analysis of CgoX mAb. Binding of DyLight-649-conjugated anti-CgoX mAb D3 to rCgoX was competed for with different concentrations of unconjugated, indicated mAbs and analysed by ELISA. Binding was determined by fluorescence measurement (Ex 646/Em 674). Data are presented as mean ± s.d. ( n = 2). f Saturation binding curve was generated by plotting absorbance signals (OD 450nm ) of increasing amounts of anti-CgoX huMAb D3 to rCgoX coated on ELISA MaxiSorp plate using the GraphPadPrism 8.4 software. Kd was calculated by non-linear fitting and the equation for one-site binding model [Y = Bmax*X/(Kd + X)].

Article Snippet: Upon washing with TBST (3 × 15 min), HRP conjugated secondary goat anti-mouse IgG (Sigma-Aldrich, Germany, cat. No. A3673) or goat anti-human IgG antibody (Bio-Rad, Germany, cat. No. 172-1050) were diluted 1:3,000 and applied for 1 h at room temperature, respectively.

Techniques: Microarray, Binding Assay, Staining, Sequencing, Enzyme-linked Immunosorbent Assay, Fluorescence, Generated, Software

Journal: NPJ Vaccines

Article Title: Epitope-specific immunity against Staphylococcus aureus coproporphyrinogen III oxidase

doi: 10.1038/s41541-020-00268-2

Figure Lengend Snippet: a Competition ELISA. Binding of anti-CgoX mAb D3 to rCgoX coated on ELISA MaxiSorp plate was competed for with CgoX-D3-BSA conjugate. Binding was detected with anti-mIgG-HRP and compared to control sample (BSA). b Anti-CgoX-BSA IgG titer. Sera of two CgoX-D3-BSA immunised mice were collected at day 68 and analysed together with preimmune serum for anti-CgoX IgGs by ELISA. c Survival of mice challenged with S. aureus upon immunisation with CgoX-D3 epitope peptide conjugated with BSA. BALB/c mice ( n = 11) immunised with CgoX-D3-BSA or the carrier protein BSA (black) as control group ( n = 10), were infected i.p. with 3.3 × 10 7 cfu S. aureus USA300 mixed with 5% mucin from porcine stomach. Significance was calculated by Log-rank (Mantel-Cox) test.

Article Snippet: Upon washing with TBST (3 × 15 min), HRP conjugated secondary goat anti-mouse IgG (Sigma-Aldrich, Germany, cat. No. A3673) or goat anti-human IgG antibody (Bio-Rad, Germany, cat. No. 172-1050) were diluted 1:3,000 and applied for 1 h at room temperature, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Control, Infection