Journal: Nucleic Acids Research

Article Title: Sequence-dependent recruitment of SRSF1 and SRSF7 to intronless lncRNA NKILA promotes nuclear export via the TREX/TAP pathway

doi: 10.1093/nar/gkab445

Figure Lengend Snippet: Functional impairment of cell migration suppression for NKILA lacking CAR-N. ( A ) Wound healing in cell migration assay in MCF-7 cells treated with TGF-β. ( B ) Quantification of cell migration at the indicated time points relative to 0 h ( n = 3), *** P < 0.001, ** P < 0.01. ( C ) Western blot showing the relative expression of E-cadherin and vimentin; β-actin is used as the loading control.

Article Snippet: Antibodies targeted to SRSF1 (ProteinTech 12929–2-AP), SRSF7 (ProteinTech 11044-1-AP), DHX9 (ProteinTech 17721-1-AP), HNRNPC (ProteinTech 11760-I-AP), PRPF8 (ProteinTech 11171-I-AP), SRSF9 (ProteinTech 17926-I-AP), U2AF2 (Santa Cruz-48804), E-cadherin (CST-14472), vimentin (ProteinTech 10366–1-AP), and β-actin (Origene-TA811000), all used at a 1:1000 dilution, and to tubulin (Sigma T9026; 1:10 000 dilution), were used for western blotting.

Techniques: Functional Assay, Migration, Cell Migration Assay, Western Blot, Expressing

Journal: Poultry Science

Article Title: Molecular cloning of duck CD40 and its immune function research

doi: 10.1016/j.psj.2021.101100

Figure Lengend Snippet: Analysis of duck CD40 tissue distribution in 5-day-old (A) and 60-day-old (B). Tissue distribution of duCD40 were detected by RT-qPCR. The mRNA expression levels were normalized to the expression of the β-actin gene. Calculation method was 2 −△△Ct .

Article Snippet: The membrane was blocked with 5% milk for 1.5 h in TBST (0.1% Tween-20 in PBS) and then incubated with mouse anti-His monoclonal antibody (Ruiyingbio, Suzhou, China) or mouse anti-β-actin monoclonal antibody (Transgen Biotech, Beijing, China) for 2 h, in 37℃.

Techniques: Quantitative RT-PCR, Expressing

Journal: Poultry Science

Article Title: Molecular cloning of duck CD40 and its immune function research

doi: 10.1016/j.psj.2021.101100

Figure Lengend Snippet: Effects of DPV and DHAV-1 on duCD40 and CD40L transcript levels in DEF. DEF cells were challenged by DPV (200μL/well) and DHAV-1 (200μL/well), at the indicated timepoints, the mRNA level of duCD40 (A, B) and CD40L (C, D) were measured by RT-qPCR. Mock group treated with equal volume of PBS. Fold change for mRNA levels was standardized by β-actin levels, data are mean ± SEM (n = 3), significant difference was indicated by asterisks, (*, P < 0.05, **, P < 0.01).

Article Snippet: The membrane was blocked with 5% milk for 1.5 h in TBST (0.1% Tween-20 in PBS) and then incubated with mouse anti-His monoclonal antibody (Ruiyingbio, Suzhou, China) or mouse anti-β-actin monoclonal antibody (Transgen Biotech, Beijing, China) for 2 h, in 37℃.

Techniques: Quantitative RT-PCR

Journal: Genes & Cancer

Article Title: Correlation between c-Met and ALDH1 contributes to the survival and tumor-sphere formation of ALDH1 positive breast cancer stem cells and predicts poor clinical outcome in breast cancer

doi: 10.18632/genesandcancer.148

Figure Lengend Snippet: A. c-Met expression in Basal-like type of breast cancer cell lines, MDA-MB157 and MDA-MB468 were analyzed by Immunoblot. β-actin was used as an internal control. B . Viability of MDA-MB157 cells after treatment with c-Met inhibitors (1, 10 and 100 μM) compared with 0.02% DMSO for 3 days was assessed by the amount of formazon formed by WST assay. Numerical values of test groups are shown with respect to 0.02% DMSO treated group. All data is represented as the mean ± S.D. of three independent experiments. C . c-Met phosphorylation level in MDA-MB157 was analyzed by immunoblot. MDA-MB157 cells were treated for 6h with Crizotinib (1.5 μM), Foretinib (1.5 μM), PHA-665752 (10 μM) and Tivantinib (0.5 μM).

Article Snippet: Mouse monoclonal β-actin antibody was obtained from Wako Inc. (Japan).

Techniques: Expressing, Western Blot, WST Assay

Journal: Genes & Cancer

Article Title: Correlation between c-Met and ALDH1 contributes to the survival and tumor-sphere formation of ALDH1 positive breast cancer stem cells and predicts poor clinical outcome in breast cancer

doi: 10.18632/genesandcancer.148

Figure Lengend Snippet: A. c-Met and Phosphorylated c-Met (p-Met) expression in ALDH1 high or ALDH1 low cells from MDA-MB157 were analyzed by Immunoblot. β-actin was used as an internal control. B.-C. Cell viability based on formation of formazon product as assessed by the WST-1 assay after 3 days of treatment with c-Met inhibitors, Crizotinib, Foretinib, PHA-665752, and Tivantinib (0.03, 0.1, 0.3, 1, 3 and 10 μM) in ALDH1 high cells derived from MDA-MB157 (B) and MDA-MB468 (C). Numerical values of test groups are shown with respect to 0.02% DMSO treated group. D. In vitro IC 50 values of c-Met inhibitors in ALDH1 high cells derived from MDA-MB 157 and MDA-MB 468. All data is represented as the mean± S.D. from three independent experiments.

Article Snippet: Mouse monoclonal β-actin antibody was obtained from Wako Inc. (Japan).

Techniques: Expressing, Western Blot, WST-1 Assay, Derivative Assay, In Vitro

Journal: Frontiers in Immunology

Article Title: The Non-Receptor Protein Tyrosine Phosphatase PTPN6 Mediates a Positive Regulatory Approach From the Interferon Regulatory Factor to the JAK/STAT Pathway in Litopenaeus vannamei

doi: 10.3389/fimmu.2022.913955

Figure Lengend Snippet: Tissue distribution and subcellular localization of LvPTPN6. (A) Expression of LvPTPN6 in L. vannamei tissues detected by qRT-PCR with EF-1α as internal control. The expression level of LvPTPN6 in pyloric cecum was set as the baseline (1.0). Each bar represents the mean ± SD ( n = 4). (B) Subcellular localization of HA-tagged LvPTPN6 was detected by confocal laser scanning microscopy analysis in S2 cells. LvPTPN6 was stained with Alexa Fluor 488 (green), the cytomembranes were visualized by β-actin stain with Alexa Flour 594 (red), and the nuclei were stained with Hoechst 33342 (blue).

Article Snippet: For nuclear translocation of L. vannamei STAT, shrimps were challenged with Poly(I:C) for 12 h as mentioned above, then hemolymph smear samples were made on siliconized slides and fixed with 4% paraformaldehyde for 10 min. For immunofluorescence assay, 4% paraformaldehyde fixed cells were infiltrated with 1% Triton X-100 for 20 min then successively incubated with rabbit Ab against HA (CST, Danvers, MA, USA) or shrimp STAT (GL Biochem, Shanghai, China) together with mouse Ab against β-actin (MBL, Tokyo, Japan) and Alexa Fluor 488-conjugated goat anti-rabbit Ab (CST, USA) together with Alexa Fluor 594-conjugated goat anti-mouse Ab (CST, USA).

Techniques: Expressing, Quantitative RT-PCR, Confocal Laser Scanning Microscopy, Staining

Journal: Frontiers in Immunology

Article Title: The Non-Receptor Protein Tyrosine Phosphatase PTPN6 Mediates a Positive Regulatory Approach From the Interferon Regulatory Factor to the JAK/STAT Pathway in Litopenaeus vannamei

doi: 10.3389/fimmu.2022.913955

Figure Lengend Snippet: Nuclear localization of STAT regulated by LvPTPN6. (A) qRT-PCR analysis of the LvPTPN6 mRNA level in hemocyte and gill at 48 h post dsRNA injection. Values in the dsRNA-GFP control group were set as the baseline (1.0). Each bar represents the mean ± SD ( n = 4), ** p < 0.01 by two-tailed unpaired Student’s t -test. (B) Western blot analysis of the protein level of STAT located in the cytoplasm and nucleus of hemocytes after dsRNA injection. (C) Immunofluorescence intensities (arbitrary units, AU) of cytoplasm- and nuclear-localized STAT in dsNRA-GFP- and dsRNA-PTPN6-treated hemocytes. Immunofluorescence intensities were calculated using JACoP with an ImageJ plugin from four randomly selected microscopic vision fields ( Supplemental Figure 3 ). * p < 0.05 by two-tailed unpaired Student’s t -test. (D) Immunofluorescent analysis of STAT in hemocytes after dsRNA injection. STAT was stained with Alexa Fluor 488 (green), the cytomembranes were visualized by β-actin stain with Alexa Flour 594 (red), and the nuclei were stained with Hoechst 33342 (blue). (E) Western blot analysis of the protein level of shrimp STAT located in the cytoplasm and nucleus of S2 cells overexpressed with LvPTPN6 or GFP (negative control). (B, E) In cytoplasm, the gray values of STAT bands were normalized to those of the cytoplasmic internal control of β-actin, and Histone H3 was detected to verify no contamination of nuclear protein. In nucleus, the gray values of STAT bans were normalized to those of the nuclear internal control of Histone H3, and β-actin was detected to verify no contamination of cytoplasmic protein. (F) Western blot analysis of the dimer and monomer levels of STAT through native PAGE after overexpressing LvPTPN6 in S2 cells. The gray values of STAT bans were normalized to those of the internal control of β-actin. (B–F) Each bar is mean ± SD of three independent quantification of the electrophoretic bands, ** p < 0.01 by two-tailed unpaired Student’s t -test.

Article Snippet: For nuclear translocation of L. vannamei STAT, shrimps were challenged with Poly(I:C) for 12 h as mentioned above, then hemolymph smear samples were made on siliconized slides and fixed with 4% paraformaldehyde for 10 min. For immunofluorescence assay, 4% paraformaldehyde fixed cells were infiltrated with 1% Triton X-100 for 20 min then successively incubated with rabbit Ab against HA (CST, Danvers, MA, USA) or shrimp STAT (GL Biochem, Shanghai, China) together with mouse Ab against β-actin (MBL, Tokyo, Japan) and Alexa Fluor 488-conjugated goat anti-rabbit Ab (CST, USA) together with Alexa Fluor 594-conjugated goat anti-mouse Ab (CST, USA).

Techniques: Quantitative RT-PCR, Injection, Two Tailed Test, Western Blot, Immunofluorescence, Staining, Negative Control, Clear Native PAGE