Journal: The Journal of Cell Biology

Article Title: Small noncoding vault RNA modulates synapse formation by amplifying MAPK signaling

doi: 10.1083/jcb.201911078

Figure Lengend Snippet: Reduction of MVP expression decreases local translational activity in neuritis. (A) Schematic diagram of compartmentalized cultures of neuronal cell bodies and neurites (axons and dendrites). The gray rectangle with a blue slit in the center indicates a cell placement device, and the orange triangle indicates the neuron-containing media. The green circles indicate a neuron. (B and C) Expression analysis of puromycin-incorporated proteins in neurites of cortical neurons. Representative images of immunoblots (B) and quantified intensities of puromycin immunoreactivity relative to MAP2C immunoreactivity normalized to the level of shControl (C; mean ± SEM, n = 3 in each group from three independent experiments). *, P < 0.05 compared with neurons expressing nontarget control shRNA by one-way ANOVA test and Tukey’s multiple comparisons test. Note that the band density of proteins becomes weakened with shRNA-mediated down-regulation of mvp expression. Puromycin-incorporated proteins were not detected in cycloheximide-treated neurites. (D and E) Representative photomicrographs of puromycin-incorporated proteins in neurites of cultured cortical neurons (stained in green; counterstained with MAP2 in red) are shown in D, and quantified levels of puromycin immunoreactivity against MAP2 immunoreactivity normalized to the level of shControl are shown in E (mean ± SEM, n = 12 in each group from three independent experiments). Scale bar, 50 µm. Significant differences from the level of shControl (*, P < 0.05) were determined by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: The antibodies used and their sources are as follows: mouse anti–β-actin antibody (622101; BioLegend); mouse anti–HA-tag antibody (MMS-101R; Covance); mouse anti–MYC-tag antibody (clone 9E10; DSHB); mouse anti-FMRP antibody (clone 7G1-1; DSHB); mouse anti–His-tag antibody (clone OGHis; MBL); mouse anti–Aurora-A antibody (clone 35C1; Abcam); rabbit anti-ERK antibody (4695; Cell Signaling); rabbit anti–phospho-ERK antibody (4377; Cell Signaling); rabbit anti-AKT antibody (9272; Cell Signaling); rabbit anti–phospho-AKT antibody (9271; Cell Signaling) ; mouse anti-PSD95 antibody (MA1-046; Thermo Fisher Scientific); guinea pig anti-PSD95 antiserum (124 014; Synaptic Systems); mouse anti–Synapsin I antibody (MAB355; Millipore); guinea pig anti-VGLUT1 antibody (AB5905; Millipore); rabbit anti-MAP2 antibody (M3696; Merck); mouse anti-phosphoserine antibody (clone 4A4; Millipore); rabbit anti-phosphothreonine antibody (clone 42H4; Cell Signaling); rabbit anti-phosphoserine antibody (2630; Cell Signaling); rabbit anti-KIF3 antibody (ab11259; Abcam); rabbit anti-KIF5 antibody (ab62104; Abcam); and mouse anti-puromycin antibody (clone 12D10; Millipore).

Techniques: Expressing, Activity Assay, Western Blot, shRNA, Cell Culture, Staining

Journal: Frontiers in Neuroanatomy

Article Title: Analysis of the expression and distribution of protein O-linked mannose β1,2- N -acetylglucosaminyltransferase 1 in the normal adult mouse brain

doi: 10.3389/fnana.2022.1043924

Figure Lengend Snippet: Primary antibodies used in this work.

Article Snippet: MAP2 , Mouse, mAb , Signalway, Shanghai, China , — , — , 1:200.

Techniques: Immunohistochemistry-IF

Journal: Frontiers in Neuroanatomy

Article Title: Analysis of the expression and distribution of protein O-linked mannose β1,2- N -acetylglucosaminyltransferase 1 in the normal adult mouse brain

doi: 10.3389/fnana.2022.1043924

Figure Lengend Snippet: Cellular localization of POMGNT1. (A–I) Dual immunostaining of POMGNT1 (red) and neuronal glial cell subtype-specific markers (green) in the coronal brain sections of the cerebral cortex, including MAP2 for mature neurons (A) , S100B for resting and activated astrocytes (B) , GFAP for activated astrocytes (C) , MBP for oligodendrocytes (D) , or Iba-1 for microglia (E) , VGLUT1 for Glutamatergic neurons (F) , and GAD65 for GABAergic neurons (G) . POMGNT1 expressed in some neuronal glial cell subtype-specific marker-positive cells, including MAP2, S100B, MBP, Iba-1, GAD65, and VGLUT1, suggesting POMGNT1 may distribute in mature neurons, astrocytes, oligodendrocytes, microglia, glutamatergic neurons, and GABAergic neurons. Nearly no POMGNT1 expressed in the GFAP-positive cells suggesting POMGNT1 may not distribute in the activated astrocytes. N = 6. Scale bars = 20 μM. (H) Quantification of the percentage of the marker labeling cells containing POMGNT1 shown in panels (A–G) . N = 6. Data are presented as the mean ± SEM; ** P < 0.01; *** P < 0.001 versus the MAP2 group (one-way ANOVA); # P < 0.05 versus the VGLUT1 group (Student’s t -test). (I) The protein expression of POMGNT1 in neurons or glial cells using in vitro culturing, including HT22, MA-c, MOPC, and BV2, was detected by Western blot. β-actin as the loading control. (J) The results of the western blot were quantified. N = 4. Data are presented as the mean ± SEM; ** P < 0.01; **** P < 0.0001 versus the HT22 group (one-way ANOVA).

Article Snippet: MAP2 , Mouse, mAb , Signalway, Shanghai, China , — , — , 1:200.

Techniques: Immunostaining, Marker, Labeling, Expressing, In Vitro, Western Blot, Control