menin n terminal epitope  (Santa Cruz Biotechnology)

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin fragments are differentially localized in neurons. ( A ) WB of mouse whole brain protein samples with menin C-terminal ((C); left panel) and N-terminal ((N); right panel) epitope antibodies (n = 3 each, representative blots), depicting full length menin (black arrow), as well as N-terminal (light grey arrow) and C-terminal (dark grey arrow) menin proteolytic fragments. ( B ) WB of subcellular fractions from mouse brain protein samples with menin C-terminal ((C); top and middle panels) and N-terminal ((N); bottom panel) epitope antibodies (n = 6, representative blots). N denotes nuclear fraction, C denotes cytoplasmic fraction, S denotes synaptic fraction. Menin localizes to the nucleus, the C-menin fragment localizes to synaptic membranes, and the N-menin fragment localizes to the cytoplasm. ( C ) As in ( B ), the markers histone H3 (HH3; nuclear marker, top panel), β-tubulin (TUB; cytoplasmic marker, middle panel), and synaptophysin (SYP; synaptic marker, bottom panel), are shown to verify the subcellular fractions. ( D ) ICC localization of menin in hippocampal cultures at DIV 7 (n = 13 images, 4 independent samples, representative image), with N-terminal (i) and C-terminal (ii) epitope antibodies, and the nuclear stain DAPI (iii), (iv) shows merged channels. α-N-terminal menin signals are restricted to the nuclear and perinuclear region, α-C-terminal menin signals also exhibit punctate localization along neurites. Scale bar, 50 μm. See also Figs and .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Marker, Staining

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin is cleaved by calpain. ( A ) ICC localization of menin in hippocampal cultures at DIV 7, cultured in the presence of 0.1% DMSO vehicle control (i–v) or 20 μM PD150606 (vi–x), a cell permeable calpain inhibitor (n = 9 images, 3 independent samples each, representative images). Cells were labeled with the nuclear stain DAPI (i,vi), menin N-terminal (ii,vii) and C-terminal (iii,viii) epitope antibodies, and a neurofilament antibody (iv,ix), (v,x) shows merged channels. Scale bar, 50 μm. ( B ) Enlarged region of interest (ROI) depicting neurite structure from the boxed region shown in Av (i–iii) and Ax (iv–vi). Scale bar, 10 μm. ( C , D ) Summary data, fluorescence intensity of the nuclear α-N-menin signal was unaffected ( C ; ROIs: 0.1% DMSO n = 67, 20 μM PD150606 n = 59), whereas the neurite α-C-menin signal was reduced upon calpain inhibition ( D ; ROIs: n = 27 each). ***Statistical significance (independent t-test), P < 0.001. See also Table and Figs and .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Cell Culture, Control, Labeling, Staining, Fluorescence, Inhibition

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: The C-terminal menin fragment colocalizes with α7 subunit-containing nAChRs at presynaptic terminals. ( A ) Super resolution image of a synaptic ROI at DIV 7 (n = 9 images, 2 independent samples, representative image), cells were labeled with α-C-terminal menin (i), α-bungarotoxin (ii; α7-nAChR), and α-synaptotagmin (iii; presynaptic marker), (iv) shows merged channels. ( B ) As in ( A ), only labeled with α-PSD-95 (iii; postsynaptic marker) (n = 10 images, 2 independent samples, representative image). Arrowheads, extrasynaptic colocalization of C-menin and α7-nAChRs; arrows, synaptic colocalization with synaptotagmin ( A ) or PSD-95 ( B ). Scale bar, 2 μm. ( C ) Summary data, incidence of colocalization of synaptotagmin (SYT; presynaptic), PSD-95 (postsynaptic), and α-bungarotoxin (BTX; α7-nAChR) puncta with C-menin. Asterisks, statistical significance (Kruskal-Wallis test); ** P < 0.01. *** P < 0.001. See also Table and Fig. .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Labeling, Marker

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin mediates subunit-specific transcriptional regulation of nAChR α5. ( A ) Live cell phase contrast ( i-iii ) and GFP fluorescence ( iv-vi ) images of untreated control (i,iv), NTC shRNA (ii,v), and MEN1 shRNA (iii,vi) lentivirus transduced hippocampal cultures at DIV 7 (n = 18 images, 6 independent samples each, representative images). Scale bar, 100 μm. ( B ) Summary data, fold change gene expression in hippocampal cultures at DIV 7, relative to untreated control, determined by qPCR (n = 6, 2 independent experiments each, triplicate replicates). MEN1 knockdown reduces nAChR α5 expression. ND, qPCR signal not detected. *Statistical significance (pair wise fixed reallocation randomization test), P < 0.05–0.001. See also Table .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Fluorescence, Control, shRNA, Gene Expression, Knockdown, Expressing

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin knockdown reduces nAChR α5 subunit expression. ( A ) ICC characterization of menin and nAChR protein expression in neuronal soma from NTC shRNA and MEN1 shRNA lentivirus transduced hippocampal cultures (n ≥ 4 images, ≥2 independent samples; see Table ; representative images, DIV 7). Untransduced neurons were GFP negative (−) and transduced neurons were GFP positive (+). The expression of menin was determined with C-terminal (i,v) and N-terminal (ii,vi) epitope antibodies, and nAChRs with a nAChR α5 antibody (iii,vii), or fluorophore-tagged α-BTX (iv,viii; α7-nAChR). ICC labels are shown in a (left panels), and GFP fluorescence is shown in b (right panels). Scale bars, 20 μm. ( B–E ) Summary data, normalized somal fluorescence intensity of α-C-terminal menin ( B ), α-N-terminal menin ( C ), α-nAChR α5 ( D ), and α-BTX ( E ), in GFP + neurons relative to GFP- neurons at DIV 3–14 (ROIs: n ≥ 12 each; see Table ). Dashed lines represent a 1:1 ratio indicating no change. Asterisks, statistical significance (independent t-test or Mann-Whitney U test); * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Knockdown, Expressing, shRNA, Fluorescence, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: C-menin mediates neurite localization of α7 subunit-containing nAChRs. ( A , B ) ICC characterization of the neurite localization of C-menin and α7-nAChRs in NTC shRNA and MEN1 shRNA lentivirus transduced hippocampal cultures ( A ; n = 18 images, 5 independent samples each; representative images, DIV 7) or hippocampal cultures maintained in the presence of 0.1% DMSO or 20 μM PD150606 ( B ; n = 12 images, 2 independent samples each; representative images, DIV 7). Cells were labeled with the menin C-terminal epitope antibody (i-ii-a) or fluorophore-tagged α-BTX (iii-iv-a), and neurites were visualized with GFP ( A i-iv-b) or neurofilament ( B i-iv-b) antibodies, (i-iv-c) shows merged channels. Scale bars, 20 μm. ( C , D ) Summary data, the α-C-menin signal in neurites was reduced upon MEN1 knockdown ( C ), which coincides with a reduction of the neurite α-BTX signal ( D ). ( E ) Summary data, reduction of C-menin abundance by calpain inhibition (see Fig. ) coincides with a reduction of the neurite α-BTX signal (ROIs: n ≥ 36 each; see Table ), indicating that the synaptic targeting of α7-nAChRs requires the menin C-terminal fragment. Asterisks, statistical significance (Mann-Whitney U test); * P < 0.05. *** P < 0.001.

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: shRNA, Labeling, Knockdown, Inhibition, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin knockdown reduces the presynaptic clustering of α7 subunit-containing nAChRs. ( A , B ) Super resolution images of C-menin, α-BTX, and SYT puncta in GFP+ synaptic ROIs from NTC shRNA ( A ) and MEN1 shRNA ( B ) lentivirus transduced hippocampal cultures at DIV 7 (n ≥ 10 images, 2 independent samples each, representative images). Cells were labeled with C-terminal menin and SYT antibodies (i–v; NTC: n = 16, MEN1 : n = 14), fluorophore-tagged α-BTX and a SYT antibody (vi–x; NTC: n = 16, MEN1 : n = 15), or α-BTX and a C-terminal menin antibody (xi–xv; NTC: n = 11, MEN1 : n = 10). (i,vi,xi) shows GFP fluorescence, (ii,xiii) shows α-C-terminal menin fluorescence, (vii,xii) shows α-BTX fluorescence, (iii,viii) shows α-SYT fluorescence, (iv,ix,xiv) shows SYT/C-menin/α-BTX merged channels, (v,x,xv) shows channels merged with GFP. Scale bar, 2 μm. ( C ) Summary data, mean number of α-C-terminal menin, α-BTX, and α-SYT puncta in super resolution images. ( D ) Summary data, incidence of colocalization of SYT/C-menin/α-BTX puncta in super resolution images. Asterisks, statistical significance (independent t-test or Mann-Whitney U test); * P < 0.05. ** P < 0.01. *** P < 0.001. See also Table .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Knockdown, shRNA, Labeling, Fluorescence, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin knockdown reduces nicotine-induced presynaptic facilitation. ( A , B ) Representative patch clamp traces recorded from GFP+ hippocampal pyramidal neurons at DIV 10–14 (n ≥ 15, 3 independent experiments, representative traces). The mean frequency and amplitude of mEPSCs was analyzed before (Pre-nicotine, 10 s; top trace) and after (Post-nicotine, 10 s; bottom trace) the nicotine pulse (10 μM, 250 ms, 10 PSI; not shown). nAChR-mediated increase in mEPSC frequency (e.g. arrow) is observed in a GFP+ neuron from NTC-shRNA expressing cultures ( A ), but is absent in a GFP+ neuron from MEN1 -shRNA expressing cultures ( B ). ( C ) Summary data, incidence of nicotine-induced presynaptic facilitation in neurons from untreated control (n = 13/19), NTC shRNA (n = 11/15; GFP+) and MEN1 shRNA (n = 5/17; GFP+) transduced hippocampal cultures (post/pre-nicotine relative mEPSC frequency ≥1 ± SEM; see Fig. and Table ). *Statistical significance (Chi-squared test), P < 0.05. ( D ) Summary data, mean frequency of mEPSCs before (pre) and after (post) the nicotine pulse in the population of neurons from untreated control (n = 13), NTC shRNA (n = 11; GFP+) and MEN1 shRNA (n = 5; GFP+) transduced cultures that exhibit nicotine-induced presynaptic facilitation. ***Statistical significance (paired t-test) , P < 0.001. ( E ) Summary data, mean amplitude of mEPSCs, as in ( D ). See also Tables – and Figs – .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Knockdown, Patch Clamp, shRNA, Expressing, Control