Journal: International Journal of Molecular Sciences

Article Title: Human Rho Guanine Nucleotide Exchange Factor 11 (ARHGEF11) Regulates Dendritic Morphogenesis

doi: 10.3390/ijms18010067

Figure Lengend Snippet: Subcellular distribution and localization of ARHGEF11 in rat cerebral cortex: the method of fractionating cerebral cortex ( A ); and fractions with antibodies against ARHGEF11, synaptophysin, and PSD-95 ( B ). ARHGEF11, synaptophysin, and PSD-95 were detected in the crude synaptosomal fractions (P2); S1 (supernate 1), S2 (supernate 2). P1 (pellet 1).

Article Snippet: The first antibodies were ARHGEF11 (Acris, 1:300), post-synaptic density protein 95 (PSD-95) (Affinity BioReagents, 1:500), and synaptophysin (Sigma-Aldrich, 1:500).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Human Rho Guanine Nucleotide Exchange Factor 11 (ARHGEF11) Regulates Dendritic Morphogenesis

doi: 10.3390/ijms18010067

Figure Lengend Snippet: Formation of ARHGEF11 and synaptic marker protein complex. The immunoprecipitation of synaptophysin and PSD-95 with ARHGEF11 (Acris) or negative control (Myc antibody): input ( A ); and immunoprecipitation ( B ). ARHGEF11 was coimmunoprecipitated with synaptophysin (38 kDa) and PSD-95 (95 kDa) in P2 fractions ( B ). Three independent experiments were conducted.

Article Snippet: The first antibodies were ARHGEF11 (Acris, 1:300), post-synaptic density protein 95 (PSD-95) (Affinity BioReagents, 1:500), and synaptophysin (Sigma-Aldrich, 1:500).

Techniques: Marker, Immunoprecipitation, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Human Rho Guanine Nucleotide Exchange Factor 11 (ARHGEF11) Regulates Dendritic Morphogenesis

doi: 10.3390/ijms18010067

Figure Lengend Snippet: Localization of endogenous ARHGEF11 in cortical primary neurons. Immunofluorescent cell staining was conducted. Expression of ARHGEF11 in primary cortical neurons at 28 day in vitro (D.I.V.): ARHGEF11 (red) located in the dendrite and dendritic spine ( A , B ); ARHGEF11 (red) was colocalized with PSD-95 (green) at the punctate structures of dendrites ( C ); and ARHGEF11 (red) was colocalized with synaptophysin (green) ( D ). Three independent experiments were conducted. Dotted rectangles indicate the area of lower figure. White arrows indicate merged spines.

Article Snippet: The first antibodies were ARHGEF11 (Acris, 1:300), post-synaptic density protein 95 (PSD-95) (Affinity BioReagents, 1:500), and synaptophysin (Sigma-Aldrich, 1:500).

Techniques: Staining, Expressing, In Vitro

Journal: Scientific Reports

Article Title: 17α Estradiol promotes plasticity of spared inputs in the adult amblyopic visual cortex

doi: 10.1038/s41598-019-55158-y

Figure Lengend Snippet: Chronic monocular deprivation decreases the size of PSD95 puncta and regulates the response to repetitive visual stimulation. ( A ) Top: Fluorescent micrographs of PSD95 (yellow, representative punctum in white circle) in V1b of binocular control (Binoc, left) and following cMD (Contra cMD, middle; Ipsi cMD, right). ROI 500 μm from surface; 28.34 μm × 28.34 μm × 40 μm, 100x mag with 3x digital zoom; maximal intensity projection (MIP; 40 × 1 μm z-steps). Bottom: Significant decrease in PSD95 immunoreactive puncta size in V1b following cMD *p < 0.001, K-S Test Contra and Ipsi relative to Binoc. No change in puncta number (inset). Males (triangles) vs. females (circles): 2-way ANOVAs size: F(1,16) = 0.29, p = 0.87; number: F(1,16) = 0.76, p = 0.14, 2-way ANOVA; Binoc n = 3 males, 3 females, cMD n = 4 males, 4 females. ( B ) Top: Fluorescent micrographs of pS831 (green, representative punctum in white circle) in V1b of binocular control (Binoc, left) and following cMD (Contra cMD, middle; Ipsi cMD, right). ROI as in 2A. Bottom: No change in pS831 immunoreactive puncta size or number (inset) in V1b following cMDed. Males (triangles) vs. females (circles): 2-way ANOVA Size: F(1,16) = 0.17, p = 0.69; Number: F(1,16) = 0.29, p = 0.87; Binoc n = 3 males, 3 females, cMD n = 4 males, 4 females. ( C ) VEPs acquired from layer 2/3 in vivo in response to single full field light flash to ipsilateral (non-deprived, red) and contralateral eye (deprived, blue) normalized to the amplitude of the first response. Contralateral eye VEPs depress more rapidly than ipsilateral eye VEPs. Inset: representative raw, single VEP waveforms in response to 3 consecutive light flashes (full field, 0.5 second 90 cd/m 2 , 0.5 second 0 cd/m 2 ) to ipsilateral (red) and contralateral (blue) eyes. Second VEP amplitude normalized to first, AVG ± SEM; Ipsi: 0.59 ± 0.13, Contra: 0.29 ± 0.02; Repeated measures ANOVA, *p < 0.001, F = 47.683, between groups, p = 0.007, F = 15.773, n = 4 subjects.

Article Snippet: The following antibodies/dilutions were used: rabbit anti-estrogen receptor α (ERα; ThermoFisher, RRID: AB_325813, 1:1000); mouse anti-estrogen receptor β (ERβ; ThermoFisher; AB_2717280, 1:1000); mouse anti-parvalbumin (PV; Millipore; RRID:AB_2174013, 1:1000); mouse anti-Post Synaptic Density 95 kd (PSD95; ThermoFisher; RRID: AB_325399, 1:200); rabbit anti-phospho- Serine 831-GluR1 (pS831; Sigma Aldrich; RRID:AB_1977218, 1:1000); followed by appropriate secondary antibodies: goat anti-rabbit and anti-mouse IgG conjugated to Alexa-488 or 647 (Life Technologies; RRID:AB_143165, RRID:AB_2535805, 1:300).

Techniques: In Vivo

Journal: Scientific Reports

Article Title: 17α Estradiol promotes plasticity of spared inputs in the adult amblyopic visual cortex

doi: 10.1038/s41598-019-55158-y

Figure Lengend Snippet: Acute 17α-estradiol treatment increases PSD95 and decreases pSer831 puncta size. ( A ) Experimental timeline. Subjects received monocular deprivation from eye opening (~P14) to adulthood (>P180). 17α-estradiol (15 μg/kg, s.c.) was delivered 30 minutes prior to eye opening. ( B ) Top: Fluorescent micrographs of PSD95 immunoreactivity (yellow, representative punctum in white circle) in V1b ± 17α estradiol contralateral (left) and ipsilateral (right) to cMD (V1b; 500 μm from surface; ROI: 28.34 μm × 28.34 μm × 40 μm, 100x mag with 3x digital zoom; MIP; 40 × 1 μm z-steps). Bottom: Cumulative distribution reveals a significant increase in PSD95 immunoreactive puncta size in V1b contralateral (left), but not ipsilateral (right) to the deprived eye, *p < 0.001, K-S Test. Males (triangles) vs. females (circles): 2-way ANOVA Contra: F(1,12) = 0.98, p = 0.34, Ipsi: F(1,12) = 0.11, p = 0.75. No change in PSD 95 immunoreactive puncta number (insets). Males (triangles) vs. females (circles): 2-way ANOVA Contra: F(1,12) = 1.84, p = 0.95, Ipsi: F(1,12) = 4.27, p = 0.06, n = 8. ( C ) Top: Fluorescent micrographs of pS831 immunoreactivity (green, representative punctum in white circle) in V1b ± 17α-estradiol contralateral (left) and ipsilateral (right) to cMD. ROI as in 4b. Bottom: Cumulative distribution reveals a significant decrease in pS831 immunoreactive puncta size in V1b contralateral (left) and ipsilateral (right) to the deprived eye, *p < 0.001, K-S Test. Males (triangles) vs. females (circles): 2-way ANOVA Contra: F(1,12) = 0.138, p = 0.72; Ipsi: F(1,12) = 0.01, p = 0.91. No change in pS831 immunoreactive puncta number (insets). Males (triangles) vs. females (circles): 2-way ANOVA Contra: F(1,12) = 1.38, p = 0.26, Ipsi: F(1,12) = 2.16, p = 0.17, n = 8.

Article Snippet: The following antibodies/dilutions were used: rabbit anti-estrogen receptor α (ERα; ThermoFisher, RRID: AB_325813, 1:1000); mouse anti-estrogen receptor β (ERβ; ThermoFisher; AB_2717280, 1:1000); mouse anti-parvalbumin (PV; Millipore; RRID:AB_2174013, 1:1000); mouse anti-Post Synaptic Density 95 kd (PSD95; ThermoFisher; RRID: AB_325399, 1:200); rabbit anti-phospho- Serine 831-GluR1 (pS831; Sigma Aldrich; RRID:AB_1977218, 1:1000); followed by appropriate secondary antibodies: goat anti-rabbit and anti-mouse IgG conjugated to Alexa-488 or 647 (Life Technologies; RRID:AB_143165, RRID:AB_2535805, 1:300).

Techniques: