Journal: Frontiers in Plant Science

Article Title: Time-Resolved Investigation of Molecular Components Involved in the Induction of NO 3 – High Affinity Transport System in Maize Roots

doi: 10.3389/fpls.2016.01657

Figure Lengend Snippet: Western blot analysis carried out on the microsomal fraction extracted from the root tissue of maize seedlings treated with NO 3 – for the indicated times. (A) Western blot analysis performed using anti-PM H + -ATPase (upper panel), anti-NRT2.1 (middle panel) or anti-NRT3.1A (lower panel) antibodies. The same amount of proteins was loaded in each lane. The protein molecular weights were estimated using the ECL TM Plex Fluorescent Rainbow Markers (GE Healthcare Life Sciences). (B) Densitometric analysis of PM H + -ATPase, NRT2.1, and NRT3.1A. Data are expressed as ratio of the corresponding 0 h sample (signals were quantified using Quantity One software, Bio-Rad). Data are the means ± SE, n = 3.

Article Snippet: The resulting mixture was treated at 37°C for 20 min. For the analysis of NRT2 and NRT3, a second buffer was used (0.05 M Tris-HCl, pH 6.8, 4% (w/v) SDS, 12% (w/v) glycerol, 2% (v/v) 2- mercaptoethanol, and 0.05% (w/v) bromophenol blue), and samples were boiled for 5 min. Five micrograms of ECL TM Plex Fluorescent Rainbow Markers (GE Healthcare Amersham TM ) were used for each SDS-PAGE analysis.

Techniques: Western Blot, Software

Journal: Frontiers in Plant Science

Article Title: Time-Resolved Investigation of Molecular Components Involved in the Induction of NO 3 – High Affinity Transport System in Maize Roots

doi: 10.3389/fpls.2016.01657

Figure Lengend Snippet: Separation of PM H + -ATPase complexes by non-denaturing Deriphat-PAGE followed by SDS-PAGE. (A) Western blot analysis performed using antibodies anti-PM H + -ATPase. The molecular weight of each protein marker (ECL Plex Fluorescent Rainbow Markers) was reported on the right side. (B) Densitometric analysis of PM H + -ATPase. Data are expressed as ratio of corresponding 0 h sample considering the sum of all three forms (signals were quantified using PDQuest TM 2-D Analysis Software, Bio-Rad). Data are the means ± SE, n = 3.

Article Snippet: The resulting mixture was treated at 37°C for 20 min. For the analysis of NRT2 and NRT3, a second buffer was used (0.05 M Tris-HCl, pH 6.8, 4% (w/v) SDS, 12% (w/v) glycerol, 2% (v/v) 2- mercaptoethanol, and 0.05% (w/v) bromophenol blue), and samples were boiled for 5 min. Five micrograms of ECL TM Plex Fluorescent Rainbow Markers (GE Healthcare Amersham TM ) were used for each SDS-PAGE analysis.

Techniques: SDS Page, Western Blot, Molecular Weight, Marker, Software

Journal: The Journal of Neuroscience

Article Title: Reduction of Detyrosinated Microtubules and Golgi Fragmentation Are Linked to Tau-Induced Degeneration in Astrocytes

doi: 10.1523/JNEUROSCI.23-33-10662.2003

Figure Lengend Snippet: Redistribution of cellular organelles in T40-AV-infected astrocytes. Double-label immunofluorescence staining with MitoTracker (Mit) for mitochondria and β-tubulin (Tub) before (A) or after (B) 8 hr of T40-AV infection, for Mit and tau (C), as well as for Mit with GFP (D) 8 hr after infection. Double-label immunofluorescence staining of calnexin (Cal, E-H), a marker for ER, and MG160 (I-N), a marker for the GA, in astrocytes before (A, E, I, K) or after (B, C, F, G, J, L-N) infection with T40-AV or with GFP-AV (D, H). Mitochondria (green) and ER (green) spread over the whole cytoplasm and reached the distal ends of MTs in astrocytes before infection (A, E) and with GFP-AV infection (D, H), whereas the mitochondria and ER accumulated in the vicinity of the nucleus 8 hr after T40-AV infection (B, C, F, G), even in the cells showing preserved MT networks (B, F) and expressing small amounts of tau (C, G). However, the collapse of the ER did not lead to changes in the expression level of calnexin (O). D has a high red pseudocolor fluorescent signal attributable to the expression of GFP. The GA was clearly stained with the anti-MG160 antibody in non-infected astrocytes (I), in which the staining appeared in the anastomotic reticulum and extended to a considerable distance (K). GA staining distinctly decreased 8 hr after T40-AV infection (J), and fragmentation of the GA occurred, as shown in higher magnification (M). This fragmentation of the GA was observed as early as 4 hr after T40-AV infection (L). Double immunostaining for MG160 (red) and tau (17026; green) indicated the relationship between the loss of GA and the expression of tau (N): cells with higher expression levels of tau lost their GA (N, arrowheads), and cells with lower expression levels of tau had some GA remaining (N, arrow). Western blot analysis showed decreased expression of MG160 (O), which is consistent with the immunofluorescence staining. Mit, MitoTracker; Cal, calnexin; Tub, tubulin. Scale bars, 10 μm.

Article Snippet: An adenovirus (AV) carrying the longest human tau isoform (T40) cDNA (T40-AV) and an AV carrying green fluorescent protein (GFP) gene (GFP-AV) driven by the human cytomegalovirus promoter were constructed using an Adeno-Quest kit (QBiogene, Quebec, Canada).

Techniques: Infection, Immunofluorescence, Staining, Marker, Expressing, Double Immunostaining, Western Blot

Journal: The Journal of Neuroscience

Article Title: Reduction of Detyrosinated Microtubules and Golgi Fragmentation Are Linked to Tau-Induced Degeneration in Astrocytes

doi: 10.1523/JNEUROSCI.23-33-10662.2003

Figure Lengend Snippet: Disruption of IF network by tau accumulation in astrocytes recapitulate features of astrocytic pathology in human tauopathies. Double-label immunofluorescence staining for vimentin (Vim) and β-tubulin (Tub) before (A) and 8 hr after (B) T40-AV infection. Similar double labeling also was performed for GFAP and Tub before (D) and 8 hr after (E) T40-AV infection. Double labeling of tau (Tau) and Vim (C) or tau and GFAP (F) after T40-AV infection, as well as after GFP-AV infection (G, H). Note the collapse of the IF network and its accumulation on the vicinity of the nucleus 8 hr after T40-AV infection, even in the cells with preserved MT networks (B,E) with relatively low tau expression levels (C,F). A similar collapse of GFAP IF networks can be detected in a brain section from a patient with PSP immunostained with PHF1 and an anti-GFAP antibody (I). Note that the PHF1-positive astrocytes have lost their GFAP-positive long processes and exhibit a disrupted GFAP IF network in which there are abnormal tau accumulations (I, inset). Scale bars: A-I, 10 μm. Transmission EM shows the collapse of the IF network. Eight hours after T40-AV infection, low-power photomicrograph shows MT bundles in the cell periphery (J, arrow), and compacted IFs are seen in the vicinity of the nucleus (data not shown) but not in the peripheral area (J, white arrowhead). Higher magnification of MT bundles (K) and IFs (L) show exclusion of IFs in K and MTs in L, respectively. Scale bars: J-L, 100 nm.

Article Snippet: An adenovirus (AV) carrying the longest human tau isoform (T40) cDNA (T40-AV) and an AV carrying green fluorescent protein (GFP) gene (GFP-AV) driven by the human cytomegalovirus promoter were constructed using an Adeno-Quest kit (QBiogene, Quebec, Canada).

Techniques: Immunofluorescence, Staining, Infection, Labeling, Expressing, Transmission Assay