Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: Respiratory Research

Article Title: Genome-wide DNA methylation analysis in lung fibroblasts co-cultured with silica-exposed alveolar macrophages

doi: 10.1186/s12931-017-0576-z

Figure Lengend Snippet: SiO 2 exposure induces collagen I and α-SMA mRNA and protein expression in co-culture system in vitro. a and b Expression of collagen I and α-SMA mRNA in fibroblast in SiO 2 exposure co-culture system, mRNA expression of SiO 2 treated groups is compared to control group. c Levels of collagen I and α-SMA protein expression in fibroblast were examined by western blotting and normalized to those of GAPDH. d , e and f Collagen I and α-SMA protein expression were measured by IHC (magnification 200×). All values represent the mean ± SD in three separate experiments. * P < 0.05 compared with control group

Article Snippet: Membranes were blocked by 5% nonfat dry milk in 1× TBS, 0.1% Tween-20 for 1 h at room temperature, then incubated at 4 °C overnight with anti-Collagen I polyclonal Ab (1:2000 dilution, Abcam), or anti-α-SMA (1:2000 dilution, Abcam) or anti-GAPDH mAb.

Techniques: Expressing, Co-Culture Assay, In Vitro, Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Loureirin B Alleviates Myocardial Ischemia/Reperfusion Injury via Inhibiting PAI-1/TGF- β 1/Smad Signaling Pathway

doi: 10.1155/2022/9128210

Figure Lengend Snippet: LrB alleviated MI/R-induced myocardial fibrosis in mice. (a) Representative images of the cardiac tissues after Masson's trichrome staining and the percentage of fibrosis were analyzed. Scar bar = 100 μ m. (b) Western blot analysis for fibronectin, collagen I, collagen III, and α -SMA in the cardiac tissues. Relative protein expression of (c) fibronectin, (d) collagen I, (e) collagen III, and (f) α -SMA. Values are expressed as mean ± SD ( n = 6). +++ p < 0.001 vs. sham group. ∗ p < 0.05, ∗∗∗ p < 0.001 vs. MI/R group.

Article Snippet: The membranes were immersed in 5% nonfat milk to block the nonspecific binding for 1 h and incubated at 4°C overnight with primary antibody for fibronectin (1 : 2000 dilution; ABclonal), collagen I (1 : 500 dilution; Affinity, Changzhou, China), collagen III (1 : 1000 dilution; Affinity), α -SMA (1 : 500 dilution; ABclonal), PAI-1 (1 : 1000 dilution; Abcam), uPA (1 : 500 dilution; Affinity), tPA (1 : 500 dilution; Affinity), TGF- β 1 (1 : 400 dilution; Affinity), TGF- β 1R (1 : 1000 dilution; Affinity), p-Smad2/3 (1 : 500 dilution; ABclonal), Smad2/3 (1 : 1000 dilution; ABclonal), and GAPDH (1 : 10000 dilution; Proteintech, Wuhan, China).

Techniques: Staining, Western Blot, Expressing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Loureirin B Alleviates Myocardial Ischemia/Reperfusion Injury via Inhibiting PAI-1/TGF- β 1/Smad Signaling Pathway

doi: 10.1155/2022/9128210

Figure Lengend Snippet: LrB inhibited Ang II-induced synthesis of fibronectin and collagen in CFs via regulating PAI-1. (a) Western blot analysis for fibronectin, collagen I, collagen III, and α -SMA in CFs. (b) Relative protein expression of fibronectin, collagen I, collagen III, and α -SMA in CFs. Values are indicated as mean ± SD ( n = 3). +++ p < 0.001 vs. control group. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. Ang II group. & p < 0.05, && p < 0.01 vs . Ang II + LrB + vector group.

Article Snippet: The membranes were immersed in 5% nonfat milk to block the nonspecific binding for 1 h and incubated at 4°C overnight with primary antibody for fibronectin (1 : 2000 dilution; ABclonal), collagen I (1 : 500 dilution; Affinity, Changzhou, China), collagen III (1 : 1000 dilution; Affinity), α -SMA (1 : 500 dilution; ABclonal), PAI-1 (1 : 1000 dilution; Abcam), uPA (1 : 500 dilution; Affinity), tPA (1 : 500 dilution; Affinity), TGF- β 1 (1 : 400 dilution; Affinity), TGF- β 1R (1 : 1000 dilution; Affinity), p-Smad2/3 (1 : 500 dilution; ABclonal), Smad2/3 (1 : 1000 dilution; ABclonal), and GAPDH (1 : 10000 dilution; Proteintech, Wuhan, China).

Techniques: Western Blot, Expressing, Control, Plasmid Preparation

Journal: eLife

Article Title: The membrane-associated proteins FCHo and SGIP are allosteric activators of the AP2 clathrin adaptor complex

doi: 10.7554/eLife.03648

Figure Lengend Snippet: In ( A ) and ( B ) APA domains from FCHo homologs were expressed as HaloTag fusions in HEK293T cells ( Ce , C. elegans ; Mm , M. musculus ). ( A ) Silver-stained gel of affinity-purified proteins following proteolytic cleavage from the HaloTag. Arrows indicate bands of presumed identity. ( B ) The top ten human proteins purified using two different APA baits, as detected by MudPIT mass spectrometry. Nonspecific proteins also found in the control were removed. Values represent the mean % distributed Normalized Spectral Abundance Factor (dNSAF × 100) from three independent experiments. The values of all four AP2 subunits were totaled to determine the amount of complex in each sample. Multiple isoforms of alpha, beta, and phosphatidylinositol 5-phosphate 4-kinase type-2 (PIP4K2) were summed. WD repeat-containing protein 48 (WDR48), epidermal growth factor receptor substrate 15 (EPS15), adaptin ear-binding coat-associated protein 2 (NECAP2), and serine/threonine-protein kinase PLK1 (PLK1). See for complete results. ( C ) APA pulldowns using bacterially expressed proteins. Purified HaloTag with (HT + APA) and without (HT) the APA domain from mouse SGIP1 were incubated with purified AP2 fragments followed by TEV protease cleavage to release the APA bait. Silver-stained gel of the eluted proteins. Note that the alpha/sigma and beta/mu hemicomplexes are soluble in our hands and that the AP2 appendage (ear) domains exhibit non-specific binding in this assay. DOI: http://dx.doi.org/10.7554/eLife.03648.017

Article Snippet: For western blot analysis of FCHo1 interactors, 20 μl of purified complexes were electrophoresed, transferred, and blotted as described above using the following primary antibodies: mouse anti-alpha adaptin (610501; 1:2000; BD Biosciences, San Jose, CA), rabbit anti-AP2B1 (151961; 1:1000; Abcam), rabbit anti AP2M1 (75995; 1:1000; Abcam) and rabbit anti-AP2S1 (128950; 1:10000; Abcam).

Techniques: Staining, Affinity Purification, Purification, Mass Spectrometry, Binding Assay, Incubation