Journal: BMC Pulmonary Medicine

Article Title: Hyperoxia but not high tidal volume contributes to ventilator-induced lung injury in healthy mice

doi: 10.1186/s12890-023-02626-x

Figure Lengend Snippet: The expression of 8-OHdG in lung tissues. A Representative confocal images (left) and quantitative data (right) of 8-OHdG-positive cells in lung tissues. B Representative blots (left) and quantitative data (right) on the expression of 8-OHdG in lungs. Data are normalized to α-tubulin and represented as the means ± SD, n = 3 ~ 5 in each group. ** p < 0.01 vs. Ctr group. V T : tidal volume. Additional file is the original WB image in the manuscript

Article Snippet: After blocking, the membranes were incubated with primary antibodies against RhoA (1:1000 dilution; cat. no. 2117s; CST), ROCK1 (1:1000 dilution; ab156284; Abcam), MLC2 (1:1000 dilution; cat. no. 3672s; CST), p-MLC2 (1:1000 dilution; cat. no. 3671s; CST), 8-OHdG (1:500 dilution; sc-393,871; Santa Cruz), or α-Tubulin (1:1000 dilution; cat. no. 3873 S; CST) overnight at 4 ̊C, respectively; followed by the appropriate horseradish peroxidase-conjugated secondary antibodies (Dako).

Techniques: Expressing

Journal: BMC Pulmonary Medicine

Article Title: Hyperoxia but not high tidal volume contributes to ventilator-induced lung injury in healthy mice

doi: 10.1186/s12890-023-02626-x

Figure Lengend Snippet: Western blot analysis on the expression of RhoA, ROCK1, MLC2, and p-MLC2 in lungs. Representative blots (left) and quantitative data (right) on the expression of RhoA ( A ), ROCK1 ( B ), MLC2 ( C ), and p-MLC2 ( D ). Data are normalized to α-tubulin and represented as the means ± SD, n = 3 ~ 5 in each group. V T : tidal volume. Additional file is the original WB image in the manuscript

Article Snippet: After blocking, the membranes were incubated with primary antibodies against RhoA (1:1000 dilution; cat. no. 2117s; CST), ROCK1 (1:1000 dilution; ab156284; Abcam), MLC2 (1:1000 dilution; cat. no. 3672s; CST), p-MLC2 (1:1000 dilution; cat. no. 3671s; CST), 8-OHdG (1:500 dilution; sc-393,871; Santa Cruz), or α-Tubulin (1:1000 dilution; cat. no. 3873 S; CST) overnight at 4 ̊C, respectively; followed by the appropriate horseradish peroxidase-conjugated secondary antibodies (Dako).

Techniques: Western Blot, Expressing

Journal: Nucleic acids research

Article Title: Nuclear AGO2 supports influenza A virus replication through type-I interferon regulation.

doi: 10.1093/nar/gkaf268

Figure Lengend Snippet: Figure 1. IAV virus NS1 induces AGO2 nuclear translocation ( A ) R epresentativ e AGO2 immunoblots from cytoplasmic (C) and nuclear (N) lysates in HEK293 and HEK293T cells. GAPDH served as cytoplasmic marker and HIST3H3 as nuclear marker. n = 3, ( B ) Representative AGO2 immunoblots from cytoplasmic (C) and nuclear (N) lysates in HEK293 cells transfected with plasmid expressing SV40 Large T antigen. HEK293T was used as a positive control. GAPDH served as cytoplasmic marker and HIST3H3 as nuclear marker. n = 3, ( C ) Representative AGO2 immunoblots from cytoplasmic (C) and nuclear (N) lysates in HEK293 cells infected with PR8 virus at MOI 0.1; 2; 10 for 16 h. GAPDH served as cytoplasmic marker and HIST3H3 as nuclear marker. n = 3, ( D ) Representatives AGO1, AGO2, and AGO3 immunoblots from cytoplasmic (C) and nuclear (N) lysates in HEK293 cells infected with PR8 virus at MOI 10 for 16 h. GAPDH and β-Tubulin served as cytoplasmic marker and HIST3H3 as nuclear marker. YBX1 served as a control for shuttling protein. n = 3, ( E ) Immunofluorescence images of AGO2 in HEK293 cells (upper panel) as well as AGO2 and PR8-mCherry in HEK293 infected with PR8-NS1-mCherry virus at MOI 10 for 16 h (lower panel). DAPI stained for DNA and Phalloidin stained for F-actin. ( F ) Representative AGO2 immunoblots from cytoplasmic (C) and nuclear (N) lysates in HEK293 cells transfected with PB1, PB2, NP, M, HA, NS1, and PA expressing plasmids for 2 da y s. G APDH serv ed as cytoplasmic mark er and HIS T3H3 as nuclear mark er . n = 3. Cartoon created using Biorender .com ( G ) R epresentativ e AGO2 and NS1 immunoblots from cytoplasmic (C) and nuclear (N) lysates in HEK293 cells transfected with WT (1–230) and deletion mutant NS1 (1–80 and 1–124) expressing plasmid for 2 days. GAPDH served as cytoplasmic marker and HIST3H3 as nuclear marker. n = 3 ( H ) Immunofluorescence images of AGO2 and NS1-mCherry in HEK293 transfected with NS1-mCherry expressing plasmids for 48 h. DAPI stained for DNA. Yellow box insert highlights a cell where AGO2 and NS1-mCherry are colocalized in the nucleus, blue box insert highlights cells that were not transfected with NS1 and AGO2 remains cytoplasmic.

Article Snippet: 3 μg of plasmid was used for transient transfection using X-tremeGENETM HP DNA Transfec- FLAG (Merck, M8823) HA (Sigma, H9658) AGO1 (CST, 5053S) AGO2 (Abcam, ab32381) AGO2 11A9 for IF (Merck, MABE253) AGO3 (CST, 5054) β-Tubulin (Proteintech, 66240–1-Ig) YB1 (Abcam, ab12148) Histone H3 (CST, 44 995) p53 (Abcam, ab1101) p53 (Santa cruz, sc-126) p53 (ThermoFisher, MA5-11296) GAPDH (Abcam, ab9485) SV40 (Santa cruz, sc-147) NS1 (ThermoFisher, PA5-32243) TNRC6A (GW182) (Santa Cruz, sc56314) mCherry (Santa cruz, sc-101 529 Ubiquitin (Santa cruz, sc-8017).

Techniques: Virus, Translocation Assay, Western Blot, Marker, Transfection, Plasmid Preparation, Expressing, Positive Control, Infection, Control, Immunofluorescence, Staining, Mutagenesis