Journal: Journal of the American Society of Nephrology : JASN

Article Title: The Extracellular Matrix Receptor Discoidin Domain Receptor 1 Regulates Collagen Transcription by Translocating to the Nucleus

doi: 10.1681/ASN.2018111160

Figure Lengend Snippet: DDR1 is upregulated in injured kidney proximal tubules. (A) Paraffin kidney sections from control and biopsy specimens from three different patients with transplant AKI (Tx-AKI) were stained with anti-DDR1 antibody. Upregulated DDR1 expression is evident in the tubules of injured kidneys. (B) Paraffin kidney sections from control or one patient with Tx-AKI were stained with anti-DDR1 antibody and Lotus tetragonolobus agglutinin (LTA, a marker of proximal tubule) and analyzed by confocal microscopy. Expression of DDR1 is evident both in the cytoplasm and in the nuclei of injured proximal tubules (arrow). (C) Orthogonal projection of confocal images of kidney sections from the patient shown in (B) was performed using the imaging program Zen (black edition). Red, DDR1; blue, DAPI. (D) Non-nuclear and nuclear fractions (20 µg/lane) from kidney cortices of wild-type mice uninjured or 3 days after ischemia-reperfusion (3d-I/R) were analyzed by western blot for levels of DDR1. (E and F) Non-nuclear DDR1 and GAPDH (E) or nuclear DDR1 and PARP1 (F) bands were quantified by densitometry. Values represent DDR1/GAPDH or DDR1/PARP1 ratio and are the mean±SD of four animals. (G) Serum-starved HK-2 cells were treated with collagen I (50 µg/ml) for the time indicated. Time 0 represents cells incubated with vehicle (20 mM acetic acid) for 60 minutes. Nuclear fractions (20 µg/lane) were analyzed by western blot for levels of DDR1. (H) Nuclear DDR1 and PARP1 bands were quantified by densitometry. Values represent DDR1/PARP1 ratio and are the mean±SD of two experiments performed in triplicate. PARP1 (nuclear marker), GAPDH, or α-tubulin (non-nuclear markers) was used to evaluate fraction purity. (I) Schematic representation of the biotinylation assay performed on HK-2 cells. See text for details. (J) Nuclear fractions of HK-2 cells biotinylated (+ biotin) and treated at 37°C with collagen I for the time indicated were analyzed for levels of DDR1 or total biotinylated proteins using HRP-avidin. Nonbiotinylated (- biotin) cells treated with collagen I for the times indicated served as control. (K) Nuclear DDR1 and PARP1 of biotinylated cells were quantified and expressed as indicated above. (L) Nuclear fractions (200 µg) of biotinylated HK-2 cells treated at 37°C with collagen I for the times indicated were immunoprecipitated using streptavidin beads. Immunoprecipitated biotinylated proteins were analyzed for levels of DDR1. Cells treated at 37°C with collagen I for the time indicated in the absence of biotinylation (- biotin) or biotinylated by kept at 4°C served as negative (background for streptavidin beads) and positive (total biotinylated DDR1) controls, respectively. (M) Nuclear biotinylated DDR1 was quantified to the Coomassie protein band shown. IP, immunoprecipitation; IB, immunoblot.

Article Snippet: For double immunostaining, paraffin sections were stained with anti-DDR1 antibody, anti-collagen IV antibody (600–401–106–0.5; Rockland), collagen I antibody (ab34710; Abcam), and anti–NMHC-IIA antibody (ab89837; Abcam), together with biotinylated Lotus tetragonolobus agglutinin (cat. B-1325; Vector Laboratories), followed by secondary antibodies conjugated to AlexaFluor 555 and Fluorescein-Streptavidin (cat. SA-5001; Vector Laboratories), and mounted using ProLong Gold Antifade Mountant with DAPI (cat. {"type":"entrez-protein","attrs":{"text":"P36931","term_id":"2506707","term_text":"P36931"}} P36931 ; Thermo Scientific).

Techniques: Staining, Expressing, Marker, Confocal Microscopy, Imaging, Western Blot, Incubation, Cell Surface Biotinylation Assay, Avidin-Biotin Assay, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Transportin-3 Facilitates Uncoating of Influenza A Virus

doi: 10.3390/ijms23084128

Figure Lengend Snippet: Knockout of TNPO3 does not affect viral attachment and the expression of sialic acid receptors. ( A – C ) The efficacy of TNPO3 KO in blocking viral attachment was assessed by an attachment assay. WT and TNPO3-KO cells were infected with HuB strain virus and incubated on ice for 1 h, followed by incubation with anti-influenza virus HA protein antibody. Then, the HA proteins were analyzed by ( A ) confocal microscopy and ( B ) analyzing the average fluorescence intensity of two independent experiments as shown in ( A ) and ( C ) flow cytometry. ( D – F ) Detection of α-2,3 sialic acid expression in WT and TNPO3-KO cells. WT cells and TNPO3-KO cells were incubated with α-2,3-linked sialic acid (MAL II) and then incubated with 10 µg/mL Cy5 Streptavidin and analyzed by ( D ) confocal microscopy and ( E) analyzing the average fluorescence intensity of two independent experiments as shown in ( D ) or ( F ) flow cytometry. WT and TNPO3-KO were stained with only Cy5-streptavidin, but not treated with lectin. Scale bar = 10 μm. ( G – I ) Detection of α-2,6 sialic acid expression in WT and TNPO3-KO cells. WT cells and TNPO3-KO cells were incubated with α-2,6-linked sialic acid (SNL) and then incubated with 10 µg/mL Cy5 Streptavidin and analyzed by ( G ) confocal microscopy and ( H ) analyzing the average fluorescence intensity of two independent experiments as shown in ( G ) or ( I ) flow cytometry. WT and TNPO3-KO were stained with only Cy5-streptavidin, but not treated with lectin. Scale bar = 10 μm.

Article Snippet: The following antibodies and reagents were used in this study: anti-GAPDH mouse monoclonal antibody (Cat NO. 60004-1-Ig, Proteintech, Wuhan, China); anti-FLAG mouse monoclonal antibody (Cat NO. F1804, Sigma, Saint Louis, MO, USA); anti-HA mouse monoclonal antibody (Cat NO. M180-3, MBL, Beijing, China); anti-Lamin A/C rabbit polyclonal antibody (Cat NO. ET7110-12, HUABIO, Hangzhou, China); anti-TNPO3 rabbit monoclonal antibody (Cat NO. ET7108-80, HUABIO, Hangzhou, China); anti-IAV NP and M1 rabbit polyclonal antibodies (Cat NO. GTX125989 and GTX125928, GeneTex, Irvine, CA, USA); fluorescein labeled affinity purified antibody to mouse IgG (H+L) (Cat NO. 5230-0427, KPL, Milford, MA, USA) and Cy3-labeled antibody to Rabbit IgG (H+L) (Cat NO. 5230-0359, KPL, Milford, MA, USA); Horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies (BF03001 and BF03008, Beijing Biodragon Inmmunotechnologies, Beijing, China); DAPI (4′, 6-diamidino-2-phenylindole) (Cat NO. C1002, Beyotime, Shanghai, China); biotinylated Sambucus nigra (SNL) and Maackia amurensis Lectin II (MAL II) lectins (Cat NO. B-1305-2 and B-1265-1, Vector Lab, Burlingame, CA, USA); Cy5-streptavidin (Cat NO. SA-1500-1, Vector Lab, Burlingame, CA, USA); anti-Flag Magnetic beads (Cat NO. HY-K0207, MCE, Shanghai, China); and anti-HA Magnetic beads (Cat NO. B26202, Bimake, Shanghai, China).

Techniques: Knock-Out, Expressing, Blocking Assay, Infection, Incubation, Confocal Microscopy, Fluorescence, Flow Cytometry, Staining